PIN1 is a new therapeutic target of craniosynostosis.

Gain-of-function mutations in fibroblast growth factor receptors (FGFRs) cause congenital skeletal anomalies, including craniosynostosis (CS), which is characterized by the premature closure of craniofacial sutures. Apert syndrome (AS) is one of the severest forms of CS, and the only treatment is surgical expansion of prematurely fused sutures in infants. Previously, we demonstrated that the prolyl isomerase peptidyl-prolyl cis-trans isomerase interacting 1 (PIN1) plays a critical role in mediating FGFR signaling and that Pin1+/- mice exhibit delayed closure of cranial sutures. In this study, using both genetic and pharmacological approaches, we tested whether PIN1 modulation could be used as a therapeutic regimen against AS. In the genetic approach, we crossbred Fgfr2S252W/+, a mouse model of AS, and Pin1+/- mice. Downregulation of Pin1 gene dosage attenuated premature cranial suture closure and other phenotypes of AS in Fgfr2S252W/+ mutant mice. In the pharmacological approach, we intraperitoneally administered juglone, a PIN1 enzyme inhibitor, to pregnant Fgfr2S252W/+ mutant mice and found that this treatment successfully interrupted fetal development of AS phenotypes. Primary cultured osteoblasts from Fgfr2S252W/+ mutant mice expressed high levels of FGFR2 downstream target genes, but this phenotype was attenuated by PIN1 inhibition. Post-translational stabilization and activation of Runt-related transcription factor 2 (RUNX2) in Fgfr2S252W/+ osteoblasts were also attenuated by PIN1 inhibition. Based on these observations, we conclude that PIN1 enzyme activity is important for FGFR2-induced RUNX2 activation and craniofacial suture morphogenesis. Moreover, these findings highlight that juglone or other PIN1 inhibitors represent viable alternatives to surgical intervention for treatment of CS and other hyperostotic diseases.

Genomic Alterations and Outcomes with VEGF-Targeted Therapy in Patients with Clear Cell Renal Cell Carcinoma.

Background: Mutations in VHL, PBRM1, SETD2, BAP1, and KDM5C are common in clear cell renal cell carcinoma (ccRCC), and presence of certain mutations has been associated with outcomes in patients with non-metastatic disease. Limited information is available regarding the correlation between genomic alterations and outcomes in patients with metastatic disease, including response to VEGF-targeted therapy. Objective: To explore correlations between mutational profiles and cancer-specific outcomes, including response to standard VEGF-targeted agents, in patients with metastatic cc RCC. Methods: A retrospective review of 105 patients with metastatic ccRCC who had received systemic therapy and had targeted next-generation sequencing of tumors was conducted. Genomic alterations were correlated to outcomes, including overall survival and time to treatment failure to VEGF-targeted therapy. Results: The most frequent mutations were detected in VHL (83%), PBRM1 (51%), SETD2 (35%), BAP1 (24%), KDM5C (16%), and TERT (14%). Time to treatment failure with VEGF-targeted therapy differed significantly by PBRM1 mutation status (p = 0.01, median 12.0 months for MT versus 6.9 months for WT) and BAP1 mutation status (p = 0.01, median 6.4 months for MT versus 11.0 months for WT). Shorter overall survival was associated with TERT mutations (p = 0.03, median 29.6 months for MT versus 52.6 months for WT) or BAP1 mutations (p = 0.02, median 28.7 months for MT versus not reached for WT). Conclusions: Genomic alterations in ccRCC tumors have prognostic implications in patients with metastatic disease. BAP1 and TERT promoter mutations may be present in higher frequency than previously thought, and based on this data, deserve further study for their association with poor prognosis.

Diagnostic Accuracy of T1-Weighted Dynamic Contrast-Enhanced-MRI and DWI-ADC for Differentiation of Glioblastoma and Primary CNS Lymphoma.

BACKGROUND AND PURPOSE: Glioblastoma and primary CNS lymphoma dictate different neurosurgical strategies; it is critical to distinguish them preoperatively. However, current imaging modalities do not effectively differentiate them. We aimed to examine the use of DWI and T1-weighted dynamic contrast-enhanced-MR imaging as potential discriminative tools. MATERIALS AND METHODS: We retrospectively reviewed 18 patients with primary CNS lymphoma and 36 matched patients with glioblastoma with pretreatment DWI and dynamic contrast-enhanced-MR imaging. VOIs were drawn around the tumor on contrast-enhanced T1WI and FLAIR images; these images were transferred onto coregistered ADC maps to obtain the ADC and onto dynamic contrast-enhanced perfusion maps to obtain the plasma volume and permeability transfer constant. Histogram analysis was performed to determine the mean and relative ADCmean and relative 90th percentile values for plasma volume and the permeability transfer constant. Nonparametric tests were used to assess differences, and receiver operating characteristic analysis was performed for optimal threshold calculations. RESULTS: The enhancing component of primary CNS lymphoma was found to have significantly lower ADCmean (1.1 × 10-3 versus 1.4 × 10-3; P < .001) and relative ADCmean (1.5 versus 1.9; P < .001) and relative 90th percentile values for plasma volume (3.7 versus 5.0; P < .05) than the enhancing component of glioblastoma, but not significantly different relative 90th percentile values for the permeability transfer constant (5.4 versus 4.4; P = .83). The nonenhancing portions of glioblastoma and primary CNS lymphoma did not differ in these parameters. On the basis of receiver operating characteristic analysis, mean ADC provided the best threshold (area under the curve = 0.83) to distinguish primary CNS lymphoma from glioblastoma, which was not improved with normalized ADC or the addition of perfusion parameters. CONCLUSIONS: ADC was superior to dynamic contrast-enhanced-MR imaging perfusion, alone or in combination, in differentiating primary CNS lymphoma from glioblastoma.

Navigation programs, are they helpful for perioperative care with thyroid cancer patients?

The purpose of this study was to develop and evaluate a navigation program for patients with thyroid cancer. The navigation program was developed following an analysis of the unmet needs of patients who underwent surgery for thyroid cancer. Ninety-nine patients in the control group received usual care, and 95 in the navigation group were managed with a navigation program during the perioperative period. The effectiveness of the navigation program was assessed by administering a questionnaire to both groups. Overall satisfaction scores were significantly higher in the navigation than in the control group (p = .025), as were satisfaction scores on the continuity of information (p < .001), the continuity of management (p = .002), the continuity of relationships with healthcare providers (p<.001), and patient empowerment (p < .001). The newly developed navigation program for patients with thyroid cancer was effective in raising satisfaction levels and in actively managing the disease during the perioperative period.

Clinical outcomes with pemetrexed-based systemic therapies in RET-rearranged lung cancers.

BACKGROUND: RET rearrangements are targetable, oncogenic lung cancer drivers. While previous series have shown durable clinical benefit with pemetrexed-based therapies in ALK- and ROS1-rearranged lung cancers, the benefits of pemetrexed-based treatments in patients with RET-rearranged lung cancers relative to other genomic subsets have not previously been explored. PATIENTS AND METHODS: A retrospective review of patients with pathologically confirmed stage IIIB/IV lung adenocarcinomas and evidence of a RET, ROS1, or ALK rearrangement, or a KRAS mutation was conducted. Patients were eligible if they received treatment with pemetrexed alone or in combination. The primary outcome of progression-free survival (PFS), and secondary outcomes of overall response rate (ORR, RECIST v1.1), time to progression (TTP), and time to treatment discontinuation were compared between RET-rearranged and groups of ROS1-rearranged, ALK-rearranged, and KRAS-mutant lung cancers. RESULTS: We evaluated 104 patients. Patients with RET-rearranged lung cancers (n = 18) had a median PFS of 19 months [95% confidence interval (CI) 12-not reached (NR)] that was comparable with patients with ROS1- (23 months, 95% CI 14-NR, n = 10) and ALK-rearranged (19 months, 95% CI 15-36, n = 36) lung cancers, and significantly improved compared with patients with KRAS-mutant lung cancers (6 months, 95% CI 5-9, P < 0.001, n = 40). ORR (45%), median TTP (20 months, 95% CI 17-NR), and median time to treatment discontinuation (21 months, 95% CI 6-NR) in patients with RET-rearranged lung cancers were not significantly different compared with patients with ALK- and ROS1-rearranged lung cancers, and improved compared with patients with KRAS-mutant lung cancers. CONCLUSION: Durable benefits with pemetrexed-based therapies in RET-rearranged lung cancers are comparable with ALK- and ROS1-rearranged lung cancers. When selecting therapies for patients with RET-rearranged lung cancers, pemetrexed-containing regimens should be considered.

Anaesthetic management of a pregnant woman with carcinoid disease.

Carcinoid tumours are neuroendocrine in origin and release vasoactive substances. Carcinoid tumours may be associated with carcinoid syndrome in 2-5% of patients and result in haemodynamic instability, bronchospasm, volume and electrolyte imbalance, and hyperglycaemia. We present the anaesthetic management of a 29-year-old parturient with metastatic carcinoid tumour. Although our patient did not ultimately develop carcinoid syndrome during the peripartum period, it was important that we used a multidisciplinary team approach, with close monitoring of her antenatal progress, and planned epidural analgesia for labour and delivery.

The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation.

Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinases (MAPKs) have been implicated in diverse cellular functions, including proliferation, migration, and survival. In this study, we examined the involvement of these kinases in osteoclast differentiation by employing specific inhibitors of the kinases. The osteoclast differentiation was assessed in three different culture systems: a coculture of mouse bone marrow cells with mouse calvarial osteoblasts, a mouse bone marrow cell culture in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and a culture of bone-resident osteoclast precursor cells driven by RANKL and M-CSF. LY294002, a specific inhibitor of PI 3-kinase, potently inhibited osteoclast differentiation in all culture systems when assessed by both tartrate-resistant acid phosphatase (TRAP) staining and dentine resorption assays. Inhibition of p38 MAPK by SB202190 resulted in a strong suppression in the exogenous RANKL dependent mouse bone marrow and bone resident precursor cell cultures. Another MAPK pathway inhibitor (PD98059), which blocks the activation of extracellular signal-regulated kinase (ERK) by inhibiting the upstream kinase MAPK-ERK kinase (MEK) 1, exerted an inhibitory effect on osteoclast differentiation only at the highest concentration tested (30 micromol/L) in many cases. Whether the signaling pathways involving these kinases are activated by RANKL was also examined. The RANKL-stimulated phosphorylation of Akt, a downstream target of PI 3-kinase, and that of ERK were observed. RANKL also stimulated the activity of p38. These results suggest that PI 3 kinase, p38, and ERK play roles in osteoclast differentiation, at least in part, by participating in RANKL signaling.

Could gut-liver function derangements cause chronic venous insufficiency?

Upregulation of adhesion molecules and neutrophil infiltration of venous valve cusps may be risk factors for chronic venous insufficiency. But studies that focus on the target organ (vein) fail to consider the influence of systemic inflammation on WBC behavior in the microcirculation. This study probes the gut-liver axis as a potential source of gut-derived oxidative stress and free radical production leading to white blood cell activation in chronic venous insufficiency. Venous hemodynamics (ambulatory venous pressure, air plethysmography, duplex) and gut-derived oxidative stress markers were studied in nine patients with chronic venous insufficiency (group I) and nine age- and sex-matched control subjects with no venous disease (group II). Group I had healed venous ulcers (class 5, CEAP) but near-normal ambulatory venous pressure, to eliminate high ambulatory venous pressure as a chronic venous insufficiency risk factor. Markers of gut-derived oxidative stress included: stool analysis; intestinal permeability; hepatic detoxification challenges with caffeine, salicylate, and acetaminophen; and urine lipid peroxides. Ambulatory venous pressure did not significantly differ (group I, 42.5 +/- 5.3 mm Hg; group II, 35.5 +/- 5.5 mm Hg; p = NS). Candida overgrowth in stool distinguished group I from group II (7/9 pts vs 1/9 pts, respectively; p = 0.015). Increased intestinal permeability (lactulose/mannitol ratio) was prevalent in both groups (group I 0.07 +/- 0.02, group II 0.17 +/- 0.08, p = NS; normal range, 0.01-0.03). Both groups showed similar incidence of elevated urine lipid peroxides (5/9 pts vs 6/9 pts, respectively; p = NS), yet group I exhibited underfunction of both sulfation (group I 16.8 +/- 2.9%, group II 43.3 +/- 11%, p<0.03; normal acetaminophen recovery 16-36%) and glucuronidation (group I 30.4 +/- 4.1%, group II 64.1 +/- 14.4%, p<0.04; normal acetaminophen recovery 27%-56%) relative to oxidative stress, perhaps an indicator of diminished antioxidant capacity in patients with chronic venous insufficiency. Gut dysbiosis (as indicated by stool yeast) and hepatic detoxification challenge pathway exhaustion may lead to subclinical, systemic inflammation and peripheral white blood cell adhesion in chronic venous insufficiency. Further exploration of the relationship between oxidative stress and venous disease is needed.

Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+) T cells.

Host immune response is known to contribute to the progression of periodontitis, and alveolar bone destruction in periodontitis is associated with enhanced osteoclast activity. Therefore, we evaluated the roles of activated lymphocyte subsets in osteoclastogenesis. Osteoclast precursors were co-cultured with activated lymphocytes (B, CD4(+) T, CD8(+) T) in the presence of either macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus soluble receptor activator of NF-kappaB ligand (sRANKL), and subsequent differentiation into active osteoclasts was evaluated by a resorption assay. The activated B and CD4(+) cells, but not CD8(+) T cells, induced osteoclast differentiation in the presence of M-CSF alone. In the presence of M-CSF and sRANKL, B cells induced the formation of small but highly active osteoclasts and increased resorption, while CD8(+) T cells profoundly suppressed osteoclastogenesis. Co-culture using an insert well or supernatant suggested that both B and CD8(+) T cells acted on osteoclasts mostly via soluble proteins. Activated B cells expressed many osteoclastogenic factors including RANKL, TNF-alpha, IL-6, MIP-1alpha, and MCP-3. CD8(+) T cells expressed a substantial amount of osteoprotegerin (OPG) along with RANKL. However, blocking antibody to OPG did not reverse the suppression by CD8(+) T cells, suggesting that other factor(s) are involved. Taken together, activated B cells promoted osteoclastogenesis, while CD8(+) T cells inhibited the osteoclast formation via direct interaction. The results imply the importance of lymphocyte subpopulations in the development of periodontitis.

The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release.

Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.

Altered expression of the fragile histidine triad gene in primary gastric adenocarcinomas.

Genomic alterations and abnormal expression of the fragile histidine triad (FHIT) gene in gastric carcinomas were examined to determine whether the FHIT gene is actually a frequent target for alteration during gastric carcinogenesis. To correlate DNA and RNA lesions of the FHIT gene with the effect on FHIT protein expression, we have investigated the FHIT gene for loss of heterozygosity (LOH), aberrant transcripts, point mutations, and protein expression in 35 gastric adenocarcinomas. Allelic loss at D3S1300 was detected in 7 of 33 (21%) informative cases. Aberrant transcripts, with deletions and/or insertions, were observed in 20 of 35 (57.1%) cases and resulted from alternative splicing through exon skipping and/or insertion of the FHIT intron 5 sequence or activation of the cryptic splice site. Point mutations were not found in the FHIT coding region but detected in noncoding exon 2, 3, 4, or 5 of eight aberrant transcripts. Significant reduction of FHIT protein expression was observed in 22 of 35 (62.9%) cases. Aberrant FHIT transcription was shown to be associated with loss of FHIT protein expression. However, aberrant FHIT transcripts themselves were not associated with any clinicopathological parameters, such as age, sex, tumor site, or clinical stage. Moreover, there was no association between the presence of LOH at D3S1300 and the expression of aberrant FHIT transcripts. Nevertheless, high frequency of aberrant FHIT transcripts, significant rate of LOH at D3S1300, and altered expression of the FHIT protein indicate that alterations of the FHIT gene can play an important role in gastric carcinogenesis.

Dissolution of poorly crystalline apatite crystals by osteoclasts determined on artificial thin-film apatite.

Poorly crystalline apatite (PCA) crystals introduced into bone tissue should be stable for a definite period before they are dissolved as a result of a host response. In this report, the dissolution of PCA crystals by the action of osteoclasts was studied on artificial thin films. These consisted of PCA crystals having similar crystallographic properties to bone crystals which were developed for assaying the osteoclast activity in vitro. The dissolution of minerals by osteoclasts decreased along with the decreased amount of labile phosphate and hydrogen phosphate domains of apatite crystals, which were caused by the crystal maturation temperature. A profound effect on mineral dissolution by pH in the culture medium was also shown. Low acidity considerably increased mineral dissolution, whereas a slight alkalinity totally blocked mineral dissolution. There was little difference in the mineral dissolution behavior of osteoclasts near the physiologic pH. In addition, it was determined whether mineral dissolution by osteoclasts was dependent on the destruction of the organic matrix. Nocodazole was introduced to inhibit the secretion of hydrolytic enzymes, and acetazolamide was added to inhibit acid production by the osteoclasts. There was no significant change as a result of nocodazole addition on mineral dissolution or by the addition of acetazolamide on degradation of collagen. These results indicate that small changes in the physicochemical properties of apatite crystals can decrease resorption by osteoclasts, which can be highly activated at low pH. These results also suggest that mineral dissolution and organic degradation by osteoclasts are self-regulating.

Heptameric ring structure of the heat-shock protein ClpB, a protein-activated ATPase in Escherichia coli.

The heat-shock protein ClpB is a protein-activated ATPase that is essential for survival of Escherichia coli at high temperatures. ClpB has also recently been suggested to function as a chaperone in reactivation of aggregated proteins. In addition, the clpB gene has been shown to contain two translational initiation sites and therefore encode two polypeptides of different size. To determine the structural organization of ClpB, the ClpB proteins were subjected to chemical cross-linking analysis and electron microscopy. The average images of the ClpB proteins with end-on orientation revealed a seven-membered, ring-shaped structure with a central cavity. Their side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since the ClpB subunit has two large regions containing consensus sequences for nucleotide binding, each layer of the ClpB heptamer appears to represent the side projection of one of the major domains arranged on a ring. In the absence of salt and ATP, the ClpB proteins showed a high tendency to form a heptamer. However, they dissociated into various species of oligomers with smaller sizes, depending on salt concentration. Above 0.2 M NaCl, the ClpB proteins behaved most likely as a monomer in the absence of ATP, but assembled into a heptamer in its presence. Furthermore, mutations of the first ATP-binding site, but not the second site, prevented the ATP-dependent oligomerization of the ClpB proteins in the presence of 0.3 M NaCl. These results indicate that ClpB has a heptameric ring-shaped structure with a central cavity and this structural organization requires ATP binding to the first nucleotide-binding site localized to the N-terminal half of the ATPase.

An archaebacterial ATPase, homologous to ATPases in the eukaryotic 26 S proteasome, activates protein breakdown by 20 S proteasomes.

In eukaryotes, the 20 S proteasome is the proteolytic core of the 26 S proteasome, which degrades ubiquitinated proteins in an ATP-dependent process. Archaebacteria lack ubiquitin and 26 S proteasomes but do contain 20 S proteasomes. Many archaebacteria, such as Methanococcus jannaschii, also contain a gene (S4) that is highly homologous to the six ATPases in the 19 S (PA700) component of the eukaryotic 26 S proteasome. To test if this putative ATPase may regulate proteasome function, we expressed it in Escherichia coli and purified the 50-kDa product as a 650-kDa complex with ATPase activity. When mixed with the well characterized 20 S proteasomes from Thermoplasma acidophilum and ATP, this complex stimulated degradation of several unfolded proteins 8-25-fold. It also stimulated proteolysis by 20 S proteasomes from another archaebacterium and mammals. This effect required ATP hydrolysis since ADP and the nonhydrolyzable analog, 5'-adenylyl beta, gamma-imidophosphate, were ineffective. CTP and to a lesser extent GTP and UTP were also hydrolyzed and also stimulated proteolysis. We therefore named this complex PAN for proteasome-activating nucleotidase. However, PAN did not promote the degradation of small peptides, which, unlike proteins, should readily diffuse into the proteasome. This ATPase complex appears to have been the evolutionary precursor of the eukaryotic 19 S complex, before the coupling of proteasome function to ubiquitination.

Terminal differentiation of normal human oral keratinocytes is associated with enhanced cellular TGF-beta and phospholipase C-gamma 1 levels and apoptotic cell death.

Subculture of primary normal human oral keratinocytes (NHOK) results in terminal differentiation, leading to cell death. To investigate whether the subculture-induced death of NHOK is due to apoptosis, we studied transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, DNA fragmentation, and expression of several apoptosis-associated genes from NHOK with different passage numbers. We also determined the effect of transforming growth factor beta1 (TGF-beta1) on the induction of apoptosis in NHOK. We were able to subculture primary NHOK up to the fifth passage, at which point cells showed morphological features of differentiation. Appearance of DNA fragmentation concurrently occurred with an increase in the number of TUNEL-positive cells with higher passage numbers. The level of cellular p53 proteins was gradually decreased by the continued passage of cells, whereas the levels of intracellular and secreted TGF-beta and phospholipase C-gamma1 (PLC-gamma1) were significantly elevated by serial subculture. Exogenous TGF-beta1 also induced differentiation and apoptosis of proliferating NHOK. These data indicate that terminal differentiation of NHOK is associated with apoptosis, which is, in part, linked to elevated cellular levels of TGF-beta and PLC-gamma1.

The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation.

Knowledge about the sizes of peptides generated by proteasomes during protein degradation is essential to fully understand their degradative mechanisms and the subsequent steps in protein turnover and generation of major histocompatibility complex class I antigenic peptides. We demonstrate here that 26 S and activated 20 S proteasomes from rabbit muscle degrade denatured, nonubiquitinated proteins in a highly processive fashion but generate different patterns of peptides (despite their containing identical proteolytic sites). With both enzymes, products range in length from 3 to 22 residues, and their abundance decreases with increasing length according to a log-normal distribution. Less than 15% of the products are the length of class I presented peptides (8 or 9 residues), and two-thirds are too short to function in antigen presentation. Surprisingly, these mammalian proteasomes, which contain two "chymotryptic," two "tryptic," and two "post-acidic" active sites, generate peptides with a similar size distribution as do archaeal 20 S proteasomes, which have 14 identical sites. Furthermore, inactivation of the "tryptic" sites altered the peptides produced without significantly affecting their size distribution. Therefore, this distribution is not determined by the number, specificity, or arrangement of the active sites (as proposed by the "molecular ruler" model); instead, we propose that proteolysis continues until products are small enough to diffuse out of the proteasomes.

Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli.

The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400-450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 degreesC, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 degreesC resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.

Conversion of normal human oral keratinocytes to tumorigenic cells is associated with the acquisition of resistance to TGF-beta.

Normal human epithelial cells cannot proliferate and undergo apoptosis in the presence of transforming growth factor-beta (TGF-beta) in vitro, but many human epidermoid cancer cells are resistant to TGF-beta. Resistance to TGF-beta may thus, in part, be responsible for uncontrolled proliferation of cancer cells. Though detailed mechanisms for the resistance of cancer cells to TGF-beta remain unknown, resistance may be due to decreased expression of TGF-beta receptors from cancer cells. To investigate this possibility, we determined the expression of TGF-beta and type II TGF-beta receptor in primary normal human oral keratinocytes (NHOK), human papillomavirus-immortalized human oral keratinocytes (HOK-16B) and two tumor cell lines derived from HOK-16B (CTHOK-16B-BaP and CTHOK-16B-DMBA). Our results show that (1) the cellular and secretory TGF-beta levels in immortalized and tumor cells were notably lower than in NHOK and (2) the level of type II TGF-beta receptor of the tested cells was similar to each other. Taken together, the conversion of NHOK to tumorigenic cells may, in part, be due to the acquisition of NHOK resistance to TGF-beta through underexpression of this cytokine.

Nickel (II)-induced apoptosis and G2/M enrichment.

Treatment with certain DNA-damaging agents induce a complex cellular response comprising pertubation of cell cycle progression and/or apoptosis on proliferating mammalian cells. Our studies were focused on the cellular effects of nickel (II) acetate, DNA-damaging agent, on Chinese hamster ovary (CHO) cells. Fragmented DNAs were examined by agarose gel electrophoresis and cell cycle was determined by DNA flow cytometry using propidium iodide fluorescence. Apparent DNA laddering was observed in cells treated with 240 microM nickel (II) and increased with a concentration-dependent manner. Treatment of nickel (II) acetate resulted in apoptosis which was accompanied by G2/M cell accumulation. Proportion of CHO cells in G2/M phase was also significantly increased in cells exposed to at least 480 microM nickel (II) from 57.7% of cells in the G0/G1 phase, 34.7% in the S phase, and 7.6% in the G2/M1 phase for 0 microM nickel (II), to 58.6%, 14.5%, and 26.9% for 640 microM nickel (II). These findings suggest that nickel (II) can modulate cellular response through some common effectors involving in both apoptotic and cell cycle regulatory pathways.

Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.