Fibronectin plays a major role in hypoxia-induced lenvatinib resistance in hepatocellular carcinoma PLC/PRF/5 cells.

Resistance to lenvatinib mesylate (LEN), a systemic chemotherapy that can be administered orally, has been a major issue for treatment of hepatocellular carcinoma (HCC). Although HCC is the tumor that most exhibits intratumoral hypoxia, which has been shown to be involved in the development of treatment resistance, there are no reports of LEN resistance in HCC treatment under hypoxia. The purpose of our study was to elucidate the mechanism of treatment resistance to LEN under hypoxia using HCC cell lines. We confirmed LEN resistance under hypoxic conditions in HCC cell lines. There was a significant increase in the IC50 value of PLC/PRF/5 cells from 13.0±0.8 µM in normoxia to 21.3±1.1 µM in hypoxia, but in HepG2 cells, the increase was not significant. To elucidate the LEN resistance mechanism of PLC/PRF/5 cells under hypoxia, we performed microarray analysis and extracted genes that are thought to be related to this mechanism. Furthermore, in-silico analysis confirmed significant changes in the extracellular matrix, and among them, FN1 encoding fibronectin was determined as the hub of the gene cluster. The expression of fibronectin in PLC/PRF/5 cells examined with immunofluorescence staining was significantly elevated in and outside of cells under hypoxia, and tended to decrease when cells were exposed to LEN under normoxia. Furthermore, the fibronectin concentration in the culture solution of PLC/PRF/5 cells examined by ELISA was 2.3 times higher under hypoxia than under normoxia under LEN(-) conditions, and 1.6 times higher under hypoxia than under normoxia under LEN(+) conditions. It is assumed that in PLC/PRF/5 cells, fibronectin is probably suppressed as an indirect effect of LEN under normoxia, but transcription factors such as HIF-1α are induced under hypoxia, thus enhancing the production of fibronectin and attenuating the effect of LEN, resulting in drug resistance. This behavior of fibronectin with LEN exposure under hypoxia is probably specific to PLC/PRF/5 cells. Further studies should verify the combined effective inhibition of fibronectin and the MAPK pathway as a promising therapeutic strategy to enhance the value of LEN in HCC treatment.

Diagnostic criteria for adult-onset leukoencephalopathy with axonal spheroids and pigmented glia due to CSF1R mutation.

BACKGROUND AND PURPOSE: To establish and validate diagnostic criteria for adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) due to colony-stimulating factor 1 receptor (CSF1R) mutation. METHODS: We developed diagnostic criteria for ALSP based on a recent analysis of the clinical characteristics of ALSP. These criteria provide 'probable' and 'possible' designations for patients who do not have a genetic diagnosis. To verify its sensitivity and specificity, we retrospectively applied our criteria to 83 ALSP cases who had CSF1R mutations (24 of these were analyzed at our institutions and the others were identified from the literature), 53 cases who had CSF1R mutation-negative leukoencephalopathies and 32 cases who had cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) with NOTCH3 mutations. RESULTS: Among the CSF1R mutation-positive cases, 50 cases (60%) were diagnosed as 'probable' and 32 (39%) were diagnosed as 'possible,' leading to a sensitivity of 99% if calculated as a ratio of the combined number of cases who fulfilled 'probable' or 'possible' to the total number of cases. With regard to specificity, 22 cases (42%) with mutation-negative leukoencephalopathies and 28 (88%) with CADASIL were correctly excluded using these criteria. CONCLUSIONS: These diagnostic criteria are very sensitive for diagnosing ALSP with sufficient specificity for differentiation from CADASIL and moderate specificity for other leukoencephalopathies. Our results suggest that these criteria are useful for the clinical diagnosis of ALSP.

Partial loss of function of colony-stimulating factor 1 receptor in a patient with white matter abnormalities.

BACKGROUND AND PURPOSE: Mutations in colony-stimulating factor 1 receptor (CSF1R) cause adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Patients with ALSP can be misdiagnosed as having acute ischemic stroke due to hyperintensity lesions on diffusion-weighted magnetic resonance imaging. Mutant CSF1R proteins identified in ALSP show a complete loss of autophosphorylation of CSF1R. METHODS: We conducted mutation screening of CSF1R in 123 patients with definite acute ischemic cerebrovascular syndrome and positive family history of stroke. The pathogenicity of identified variants was evaluated using functional analyses. The levels of autophosphorylation of CSF1R in response to treatment with ligands of CSF1R were examined in cells transfected with wild-type and mutant CSF1R. RESULTS: We identified eight CSF1R variants, six were known non-pathogenic polymorphisms, whereas the other two were missense variants inducing substitution of amino acid residues (p.Glu573Lys and p.Gly747Arg). Functional assay showed that the levels of autophosphorylation of p.Gly747Arg were similar to those of wild-type when treated with ligands. The autophosphorylation of p.Glu573Lys was detectable, but significantly decreased compared with those of wild-type CSF1R (P < 0.001, two-way anova with Bonferroni). The clinical presentation of the patient with p.Glu573Lys was consistent with cerebral embolism. The patient did not have typical clinical findings of ALSP. However, periventricular white matter abnormalities, unrelated to the recent infarct, were evident on brain magnetic resonance imaging. CONCLUSIONS: In contrast to ALSP-associated missense mutations, CSF1R p.Glu573Lys variant in a patient with acute ischemic cerebrovascular syndrome showed a partial loss of autophosphorylation of CSF1R; its clinical significance warrants further investigation.

Clinical and genetic characterization of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia associated with CSF1R mutation.

BACKGROUND AND PURPOSE: The clinical characteristics of colony stimulating factor 1 receptor (CSF1R) related adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) have been only partially elucidated. METHODS: Clinical data from CSF1R mutation carriers who had been seen at our institutions or reported elsewhere were collected and analysed using a specific investigation sheet to standardize the data. RESULTS: In all, 122 cases from 90 families with CSF1R mutations were identified. The mean age of onset was 43 years (range 18-78 years), the mean age at death was 53 years (range 23-84 years) and the mean disease duration was 6.8 years (range 1-29 years). Women had a significantly younger age of onset than men (40 vs. 47 years, P = 0.0006, 95% confidence interval 3.158-11.177). There was an age-dependent penetrance that was significantly different between the sexes (P = 0.0013). Motor dysfunctions were the most frequent initial symptom in women whose diseases began in their 20s. Thinning of the corpus callosum, abnormal signalling in pyramidal tracts, diffusion-restricted lesions and calcifications in the white matter were characteristic imaging findings of ALSP. The calcifications were more frequently reported in our case series than in the literature (54% vs. 3%). Seventy-nine per cent of the mutations were located in the distal part of the tyrosine kinase domain of CSF1R (102 cases). There were no apparent phenotype-genotype correlations. CONCLUSIONS: The characteristics of ALSP were clarified. The phenotype of ALSP caused by CSF1R mutations is affected by sex.

An efficient approach to gem-difluorocyclopropylstannanes via highly regio- and stereoselective hydrostannylation of gem-difluorocyclopropenes and their unique ring-opening reaction to afford ß-fluoroallylic alcohols.

Treatment of various gem-difluorocyclopropenes with 1.2 equiv. of n-Bu3SnH in the presence of 20 mol% of Et3B at 80 °C for 4 h led to the quantitative formation of the hydrostannylated products in a highly regio- and trans-selective manner. Additionally, the prepared trans-gem-difluorocyclopropylstannanes were treated with 1.5 equiv. of MeLi in THF at -78 °C for 5 min, followed by quenching the reaction with various agents, such as H2O, alcohols, carboxylic acids, and tosylamide, to give the corresponding ß-fluoroallylic alcohols, ethers, esters, and amides respectively with exclusive Z selectivity in acceptable yields.

Multi-L1(0) domain CoPt and FePt nanoparticles revealed by electron microscopy.

The atomic structure of CoPt and FePt nanoparticles (with a diameter between 2 and 5 nm) has been studied by transmission electron microscopy. The particles have been produced by a laser vaporization cluster source and annealed under vacuum in order to promote chemical ordering. For both alloys, we observe a coexistence of crystalline and multiply twinned particles with decahedral or icosahedral shapes. In addition to particles corresponding to a single L1(0) ordered domain, we put into evidence that even small particles can display several L1(0) domains. In particular, the chemical order can be preserved across twin boundaries which can give rise to spectacular chemically ordered decahedral particles made of five L1(0) domains. The stability of such structures, which had been recently predicted from theoretical simulations, is thus unambiguously experimentally confirmed.

Low temperature ferromagnetism in chemically ordered FeRh nanocrystals.

In sharp contrast to previous studies on FeRh bulk, thin films, and nanoparticles, we report the persistence of ferromagnetic order down to 3 K for size-selected 3.3 nm diameter nanocrystals embedded into an amorphous carbon matrix. The annealed nanoparticles have a B2 structure with alternating atomic Fe and Rh layers. X-ray magnetic dichroism and superconducting quantum interference device measurements demonstrate ferromagnetic alignment of the Fe and Rh magnetic moments of 3 and 1µ(B), respectively. The ferromagnetic order is ascribed to the finite-size induced structural relaxation observed in extended x-ray absorption spectroscopy.

Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT.

We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain-containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

Conversion of 12-membered d3- and l3-CoCd metallorings into a 24-membered d3l3-CoCd metalloring.

The treatment of [Co(d- or l-ebp)]- with CdX2 (X = Br-, I-) gave a cyclic CoIII3CdII3 complex with a 12-membered metalloring, [Cd3X3{Co(d- or l-ebp)}3] (d3- or l3-1X). The use of a 1 : 1 mixture of [Co(d-ebp)]- and [Co(l-ebp)]-, instead of [Co(d- or l-ebp)]-, led to the creation of a cyclic CoIII6CdII6 complex with a 24-membered metalloring, [Cd6X6(H2O)6{Co(d-ebp)}3{Co(l-ebp)}3] (d3l3-2X). Compounds d3l3-2X were also produced when d3-1X and l3-1X were mixed in water in a 1 : 1 ratio, illustrating the conversion of a pair of homochiral metallorings into a double-sized heterochiral metalloring.

Discovery of metaflumizone, a novel semicarbazone insecticide.

Metaflumizone, (EZ)-2'-[2-(4-cyanophenyl)-1-(alpha,alpha,alpha-trifluoro-m-tolyl)ethylidene]-4-(trifluoromethoxy) carbanilohydrazide, was discovered by Nihon Nohyaku in the early 1990s and belongs to the new class of semicarbazone insecticides. It is now being globally co-developed as the animal health product, ProMeris((R)), in cooperation with Fort Dodge Animal Health and as an agricultural and consumer insecticide in cooperation with BASF. Metaflumizone was developed in a synthesis program initiated from a pyrazoline insecticide lead. In this paper, we describe the development, discovery and structure activity relationships for metaflumizone and related compounds.

Diagnostic Value of Brain Calcifications in Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia is a rare neurodegenerative disease resulting from mutations in the colony stimulating factor 1 receptor gene. Accurate diagnosis can be difficult because the associated clinical and MR imaging findings are nonspecific. We present 9 cases with intracranial calcifications distributed in 2 brain regions: the frontal white matter adjacent to the anterior horns of the lateral ventricles and the parietal subcortical white matter. Thin-section (1-mm) CT scans are particularly helpful in detection due to the small size of the calcifications. These calcifications had a symmetric "stepping stone appearance" in the frontal pericallosal regions, which was clearly visible on reconstructed sagittal CT images. Intrafamilial variability was seen in 2 of the families, and calcifications were seen at birth in a single individual. These characteristic calcification patterns may assist in making a correct diagnosis and may contribute to understanding of the pathogenesis of leukoencephalopathy.

Adenosine A2A receptor mediated protective effect of 2-(6-cyano-1-hexyn-1-yl)adenosine on retinal ischaemia/reperfusion damage in rats.

AIMS: To determine the effect of 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado), an adenosine A2A receptor agonist, on retinal ischaemia/reperfusion damage in rats. METHODS: Retinal ischaemia/reperfusion damage was induced by elevating the intraocular pressure of one eye to 130 mm Hg for 60 minutes and returning it to normal. 7 days later, retinal ischaemia/reperfusion damage was histologically quantified by measuring the thickness of retinal layers. Intraocular pressure was measured by pressure transducer. RESULTS: Retinal ischaemia/reperfusion caused cell loss in the ganglion cell layer and thinning of the inner plexiform and nuclear layer. Both ocular topical and intravenous administration of 2-CN-Ado caused a reduction of retinal ischaemia/reperfusion damage. A selective A2A receptor antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine (CSC), but not a selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or a selective A2B receptor antagonist, alloxazine, reduced the protective effect of 2-CN-Ado. While ocular topical administration of 2-CN-Ado caused a sustained reduction of intraocular pressure, intravenous administration of 2-CN-Ado showed a transient ocular hypotensive effect. CONCLUSIONS: These results suggest that 2-CN-Ado attenuates retinal ischaemia/reperfusion damage, and at least some of this protective effect of 2-CN-Ado might be mediated via activation of the adenosine A2A receptor.

Gene expressions of canine angiopoietin-1 and -2 in normal tissues and spontaneous tumours.

The angiopoietin (Ang) family of proteins are central to the regulation of angiogenesis. The purposes of this study were to determine cDNA sequences of canine Ang-1 and Ang-2 and investigate their expressions in normal tissues and spontaneous tumours. The cDNA sequences of canine Ang-1 and Ang-2 were 1,494 and 1,488 bp, and the deduced amino acid sequences were 497 and 495 residues, respectively. The cDNA sequences of canine Ang-1 and Ang-2 showed high homology with those of the other mammalian species. Canine Ang-1 and Ang-2 mRNA were detectable in all 22 normal tissues and spontaneous tumours. Higher mRNA expression level of canine Ang-2 was demonstrated in mammary simple carcinomas, haemangiosarcoma and hepatocellular carcinoma in comparison with normal tissues.

Use of oily contrast medium for selective drug targeting to tumor: enhanced therapeutic effect and X-ray image.

Highly malignant rabbit tumor (VX-2) was implanted at the periphery of the liver in 63 rabbits. Selective delivery of the anticancer agent copoly(styrenemaleic acid) conjugated to neocarzinostatin (SMANCS), which was dissolved in an oil contrast medium (Lipiodol), was performed by injection via the proper hepatic artery. The anticancer effect was also evaluated by various parameters. By using low-kVp X-ray examination of the resected rabbit liver, Lipiodol was found to distribute throughout the entire liver arterial lumina and was retained there for about 24 hr, but disappeared from the normal liver arterial lumina gradually. However, Lipiodol was retained in the tumor tissue and vessels for at least 7 days, whereas it was undetectable in any other organs. A radioactive analogue of Lipiodol, a chloroiodinated fatty acid, was prepared by using [14C]linoleic acid. This analogue was used in the study of the distribution by low-kVp X-ray examination, Sudan III staining, and autoradiography. Lipiodol remained in the tumor vessels as well as the tumor cells. The use of the radioisotope yielded a quantitative profile of Lipiodol accumulation in tumor tissues; approximately 1000 times more at 15 min and 100 times more at 3 days after the injection than that of most other organs or plasma. Its major excretion route appeared to be through the bile and then the feces. The biological activity of SMANCS was also determined and was found to be significant in both tumor and liver even 7 days after injection. No activity was found in any other organ or tissue. The relatively high biological activity of SMANCS in the nontumorous liver adjacent to the tumor may be the result of continuous drug release from SMANCS-Lipiodol in the tumor tissue. By histological examination, massive tumor necrosis and infiltration of the inflammatory cells were found in the rabbits treated with SMANCS-Lipiodol. In the rabbits treated with Lipiodol alone, necrosis of the tumor was only minimal, and no infiltration of inflammatory cells was observed. Survival periods of the treated rabbits (n = 14) were significantly longer than those of controls (n = 10); 23.1 +/- 5.5 (S.D.) days versus 16.1 +/- 2.9 days (p less than 0.005), respectively, even though only one injection was used for this highly malignant tumor. Mean tumor size for both groups at laparotomy was 163.3 +/- 83.0 sq mm and 160.5 +/- 76.5 sq mm, respectively (not significant).(ABSTRACT TRUNCATED AT 400 WORDS)

Reduction of hepatic metastases in rabbits by administration of an oily anticancer agent into the portal vein.

We studied a prophylactic chemotherapy against hepatic metastases arising from the shedding of tumor cells into the portal circulation. The therapy was done with a lymphographic oily contrast medium, Lipiodol, and a high molecular weight anticancer agent named poly(styrene-maleic acid) copolymer conjugated neocarzinostatin (SMANCS), developed in our laboratory. SMANCS was dissolved in Lipiodol by sonication (SMANCS/Lipiodol, 1 mg of SMANCS in 1 ml of Lipiodol). Twelve rabbits were simply inoculated with the highly malignant carcinoma VX-2. Fifteen rabbits were given injections of SMANCS in glucose and Lipiodol into the portal vein and were subsequently inoculated with the tumor cells. Eighteen were given injections of SMANCS/Lipiodol and then the tumor cells. These rabbits were killed 12 days later. Thirteen were given injections of the tumor cells alone and were allowed to survive. Sixteen were given injections of SMANCS/Lipiodol and then with the tumor cells; they were allowed to survive. Rabbits given injections of SMANCS/Lipiodol before tumor inoculation had significantly fewer (P less than 0.001) metastases than those not treated or those given SMANCS in glucose and Lipiodol. Survival was significantly longer [P less than 0.005; 36.0 +/- 7.7 (SD) days] with SMANCS/Lipiodol before tumor inoculation than without treatment [23.5 +/- 3.0 days]. SMANCS/Lipiodol has a prolonged anticancer effect because it remains in the portal vein and allows sustained drug release from the oil (Lipiodol) to aqueous spaces. Hepatic metastases might be prevented by portal administration of the appropriate oily anticancer agent.

Antitumor effects of SMANCS on rat mammary tumor induced by 7,12-dimethylbenz[a]anthracene.

We previously found that a high-molecular-weight anticancer agent, polystyrene-co-maleic acid conjugated neocarzinostatin (SMANCS), in which two chains of styrene/maleic acid copolymer are conjugated to the anticancer protein neocarzinostatin (NCS), accumulated more selectively in tumor tissue than in normal tissue and was more stable than NCS in blood. These results indicate that SMANCS should have less systemic toxicity and a better therapeutic effect than NCS. In this study, the antitumor activity and adverse effects of SMANCS were compared with those of NCS by using rat mammary tumor induced by 7,12-dimethylbenz[a]anthracene. When tumors of rats, that had received 7,12-dimethylbenz[a]anthracene (20 mg/kg, one dose, p.o. in oily formulation), became palpable usually after 4-20 weeks, SMANCS treatment was initiated. Thirty days after i.v. administration of SMANCS (0.1 mg/kg 3 times and 0.3 mg/kg 3 times), tumors had shrunk in 35 of 37 rats (a mean weight was about 10% of control value; or decreased to about 30% of the value of before treatment in tumor weight); tumor size had not changed in 1 rat, and in the remaining 1 rat the tumor had enlarged. Thirty days after i.v. administration of NCS, tumors had shrunk in 8 of 14 rats, but the tumor size was unchanged in 1 rat and was enlarged in 5. In the control group, all tumors had enlarged. Development of new tumors was completely prevented by the administration of SMANCS. Histological examination of sequential slices of tumor revealed clear finding of degeneration and tumor encapsulation at 30 days after initial administration of SMANCS, with an accompanying fatty degeneration, but these effects were not observed for tumors treated with NCS. Although red blood cell counts and hemoglobin amounts decreased significantly in rats receiving NCS, no such effects were apparent in the SMANCS group.

Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester.

Suspension cultures of a human monocytic leukemia cell line, THP-1, were treated with 0.16 to 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). In an original cell line, THP-1-O, cultured again from -80 degrees cryopreservation, more than 80% of the cells adhered to the glass substrate with marked morphological change within 3 hr of TPA treatment. Adherent cells became flat and amoeboid in shape, and many microvilli and flaps of the cell surface disappeared. Well-developed Golgi apparatus, rough endoplasmic reticula, and a large amount of free ribosomes were seen in the cytoplasm. On the other hand, in THP-1-R cells cultured continuously without cryopreservation for 26 months, approximately 80% of the cells adhered to the substrate 48 hr after TPA treatment. Round and ovoid shapes were kept in THP-1-R cells treated with TPA. Surface Fc receptors for immunoglobulin G were present on more than 90% of THP-1-O and THP-1-R cells and were little affected by treatment with TPA. Sixty to 70% of the TPA-treated THP-1-O and THP-1-R cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. Less than 20% of the untreated THP-1 cells were able to phagocytize yeasts and immunoglobulin G-coated sheep erythrocytes. In histochemical staining, alpha-naphthyl butyrate esterase was enhanced after treatment with TPA. Lysozyme activity in culture supernatants was not affected by TPA treatment. When exposed to latex beads and TPA, increased 14CO2 production from [1-14C]glucose in THP-1-O cells was observed. These results indicate that, after treatment with TPA, human monocytic leukemia cells may be converted into mature cells with functions of macrophages.

Transition state of an unfolding step in human cyanomet myoglobin.

We studied an unfolding step of cyanomet myoglobin (Mb) unfolding, for demonstrating dynamical structural changes in the transition state of the process. Three leucine-->alanine mutant Mbs (L29A, L72A and L104A) were prepared for this study. The urea-induced largely monophasic process was monitored by absorption spectroscopy. Linear relations between [urea] and the activation energy (delta G not equal to) of the relaxation for all the Mbs showed that the slope m not equal to urea (= delta(delta G not equal to)/delta[urea])) was altered by either reduction of pH or the L-->A mutations. Thermodynamic interpretations of the changes in m not equal to urea led to a conclusion that the exposed surface area of Mb in the transition state was determined by both protein-core stability and pH conditions. We also performed urea- and acid-denaturation experiments, and gave some inspections on differences between mutational effects on the structure of the transition state and the denatured state.

A new alternative method to quantify residual structure in 'unfolded' proteins.

Pig (pCSD1) and human (hCSD1) calpastatin domain 1 proteins were studied to characterize common features of the denatured state of proteins. These proteins were chosen for the present investigation, because pCSD1 was suggested previously to be unstructured in water even at 25 degrees C (1) [T. Konno et al., Biochim. Biophys. Acta 1342 (1997) 73-82]. hCSD1 could be expected to exhibit similar features on the basis of preliminary spectroscopic studies. In the present study, the experimental grounds for the estimate of residual structure in the unfolded state were differential scanning calorimetry heat capacity and circular dichroism (CD) measurements over the temperature range 10-80 degrees C. At selected temperatures, we studied also the effect of guanidinium hydrochloride (GdnHCl) which is known to promote further unfolding of the polypeptide chain. All other measurements were performed at pH 6 in pure water. The present results support the conclusion that the comparison of the experimentally obtained heat capacity data with theoretical heat capacity values calculated on the basis of a newly established increment system gives insight into the degree of hydration of the unfolded polypeptide chain. The percentage by which the experimental heat capacity of the unfolded polypeptide chain differs from the calculated heat capacity permits a quantitative estimate of the residual structure. This estimate is in good agreement with that based on CD absorption. The heat capacity approach has the advantage of comparing fully hydrated and partially hydrated residues in the same aqueous environment, whereas for example spectroscopic measurements, such as CD, are generally referred to the fully unfolded chain in concentrated urea or GdnHCl solutions. As the unfolded chains of pCSD1 and hCSD1 exhibit a smaller heat capacity than that calculated on the new peptide-based increment system [M. Häckel et al., J. Mol. Biol. 291 (1999) 197-213], we conclude that the residues in the unfolded polypeptide chain are less hydrated than the same residues in oligopeptides. This suboptimal hydration is the result of residual structure in the chain as observed in both CD and heat capacity measurements.

A circular dichroism study of preferential hydration and alcohol effects on a denatured protein, pig calpastatin domain I.

Pig calpastatin domain I (CSD1), a proteinase inhibitor that specifically blocks activity of calpain I and II, is a good candidate protein for studying conformational variations in the denatured form of protein. An extensive structural characterization of CSD1 reported in the first part of this work has shown that CSD1 at neutral pH is in an expanded and flexible conformation without secondary and tertiary structures. Next, we further studied cosolvent effects of protein-stabilizers, polyols and sulfate salts, as well as protein-destabilizers, alcohols, on the conformation of CSD1 monitored by far- and near-UV CD spectroscopy. We found that both groups of cosolvents at high concentration induce highly helical structures of CSD1, but without specific tertiary interactions. Based on the results on the polyols and the sulfate salts, we have suggested that the preferential hydration is one of the thermodynamic forces to induce secondary structures in the denatured state of protein. Variations in isodichroic points of changes in far-UV CD spectrum as functions of cosolvent species and their concentration have exhibited complexity of the processes. The present study implies that protein stability in the presence of cosolvents is often determined by free energy difference between the folded and the highly structured denatured state, not between the folded and the random state.