Halting hyaluronidase activity with hyaluronan-based nanohydrogels: development of versatile injectable formulations.

Hyaluronan (HA) is among the most used biopolymers for viscosupplementation and dermocosmetics. However, the current injectable HA-based formulations present relevant limitations: I) unmodified HA is quickly degraded by endogenous hyaluronidases (HAase), resulting in short lasting properties; II) cross-linked HA, although shows enhanced stability against HAase, often contains toxic chemical cross-linkers. As such, herein, we present biocompatible self-assembled hyaluronan-cholesterol nanohydrogels (HA-CH NHs) able to bind to HAase and inhibit the enzyme activity in vitro, more efficiently than currently marketed HA-based cross-linked formulations (e.g. Jonexa™). HA-CH NHs inhibit HAase through a mixed mechanism, by which NHs bind to HAase with an affinity constant 7-fold higher than that of native HA. Similar NHs, based on gellan-CH, evidenced no binding to HAase, neither inhibition of the enzyme activity, suggesting this effect might be due to the specific binding of HA-CH to the active site of the enzyme. Therefore, HA-CH NHs were engineered into injectable hybrid HA mixtures or physical hydrogels, able to halt the enzymatic degradation of HA.

Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with mass spectrometry detection.

The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening toward PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of frontal affinity chromatography coupled to mass spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments toward new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes.

Synthesis, characterization and biological evaluation of ureidofibrate-like derivatives endowed with peroxisome proliferator-activated receptor activity.

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Quaternary structure of Dictyostelium discoideum nucleoside diphosphate kinase counteracts the tendency of monomers to form a molten globule.

Multimeric enzymes that lose their quaternary structure often cease to be catalytically competent. In these cases, conformational stability depends on contacts between subunits, and minor mutations affecting the surface of the monomers may affect overall stability. This effect may be sensitive to pH, temperature, or solvent composition. We investigated the role of oligomeric structure in protein stability by heat and chemical denaturation of hexameric nucleoside diphosphate kinase from Dictyostelium discoideum and its P105G mutant over a wide range of pH. The wild-type enzyme has been reported to unfold without prior dissociation into monomers, whereas monomer unfolding follows dissociation for the P105G mutant (Giartosio et al. (1996) J. Biol. Chem. 271, 17845-51). We show here that these features are also preserved at alkaline pH, with the wild-type enzyme always hexameric at room temperature whereas the mutant dissociates into monomers at pH >or=10. In acidic conditions (pH

Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study.

The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications.

Spontaneous cerebellar hemorrhage: clinical remarks on 50 cases.

BACKGROUND: Only during the past 10 years have spontaneous cerebellar hemorrhages became a well-defined nosological entity. The surgical indication remains debatable. Our primary objective in this study was to set the criteria for undertaking surgery by determining the critical diameter of the hematoma and considering the patients' neurological status (Glasgow Coma Scale). METHODS: During the 8-year period 1990 through 1997 a series of 50 consecutive patients with spontaneous cerebellar hemorrhage were admitted to the Emergency Neurosurgery Unit, University of Rome "La Sapienza" (Italy). On admission all patients underwent a standard neurological examination, (Glasgow Coma Scale) and a computed tomographic scan. The diameter and the site of the hematoma, a coexisting tight posterior fossa, and the presence of hypertensive hydrocephalus were the criteria, in association with the patients' neurological status, used as indications for surgery. RESULTS: Operative mortality was nil; and perioperative mortality eight patients (16%, increasing to 24% including the four patients who were deeply comatose on admission). Most patients who died (seven of eight) had two or more general medical risk factors (arterial hypertension and diabetes mellitus; arterial hypertension and liver disease; or liver disease and hematological disorders). CONCLUSION: In patients presenting with spontaneous cerebellar hemorrhage the essential criteria indicating surgery are a hematoma 40 mm x 30 mm on CT imaging in the cerebellar hemisphere or 35 mm x 25 mm on CT imaging in the vermis, the presence of a tight posterior fossa (critical size reduced by 10 mm), and a Glasgow Coma Score less than 13.

DNA-protein cross-linking in nuclei of immature and mature chicken erythrocytes.

DNA-protein cross-linkages were formed in isolated nuclei from immature and mature chicken erythrocytes by reaction with cis-diammine dichloroplatinum. On the basis of electrophoretic behaviour, the most abundant proteins involved in the cross-linking appeared to be present also in preparations of nuclear matrix. The maturation of the erythrocyte, which is accompanied by transcriptional inactivation, leads to a decrease in the amount of DNA-interacting proteins, to a loss of proteins capable of a specific recognition of DNA sequences and, unexpectedly, to the appearence of some new DNA-protein interactions. At least three cross-linked proteins were found predominantly or exclusively in nuclei of immature cells, and three others in those of mature ones. The three DNA-bound proteins, typical of mature erythrocytes, were not found among the components of a high-salt preparation of nuclear matrix. The results obtained suggest that, in addition to the well-known histone H5 and MENT protein, these newly identified DNA-bound proteins contribute to the formation of the condensed, inactive chromatin characteristic of mature erythrocyte.

Medulloblastoma in late adults. Case report and critical review of the literature.

BACKGROUND: Medulloblastoma in late adults (older than 65 years) is an exceptional occurrence; in fact only 8 cases are reported with complete clinical notes in the literature. METHODS: The authors describe a case of medulloblastoma occurring in a 68-year-old man and analyzed cases reported in the literature. RESULTS: The overall average age of the patients was 72.8 years (range 67-88 years), and the male prevalence of this tumour (70% of cases) seems to be unrelated to age. There is a lateral predominance in late adults (77.7% of cases). Seven patients underwent operation and subsequently these patients underwent a course of radiation therapy. Only two patient was treated with postoperative chemotherapy. Median survival for seven patients treated was 43.2 months (range 23-96 months). CONCLUSIONS: It is interesting to note that: 1) histological analysis revealed a classic type medulloblastoma (88.8% of cases) similar to the children: 2) site of the tumor is lateral similar to the adults (77.7% of cases).

An asymptomatic hypertrophic pacchionian granulation simulating osteolytic lesion of the calvaria.

Osteolytic lesions can be seen in various diseases. We present a rare case of symptomatic hypertrophic pacchionian granulation mimicking bone tumor in the calvaria. A 50-year-old woman suffered from a previous VII cranial nerve peripheral paresis accompanied by headache. A plain radiograph revealed a punched-out paramedial occipital lesion. Precontrast-enhanced computed tomographic scans demonstrated a hypodense mass, with a defect of both tables of the left occipital bone. Magnetic resonance imaging (MRI) demonstrated a hypointense mass on the T1-weighted image and isointense to cerebrospinal fluid on the T2-weighted image, with capsule-like contrast enhancement by gadolinium. A biopsy was performed. Histologically, hypertrophic pacchionian granulation was diagnosed. The patient has had no growth for 2 years. This case suggests the need to include hypertrophic pacchionian granulation in the differential diagnosis of punched-out lesions.

Cerebral metastasis from ovarian carcinoma. Some observations about treatment.

Systemic metastases from ovarian carcinoma are frequent, but they rarely affect the central nervous system. The treatment of this type of metastases in not clear. Two cases of solitary cerebral metastasis from ovarian carcinoma are reported. Two patients, submitted to therapeutic protocol established for ovarian carcinoma, presented after 17 and 25 months respectively the appearance of symptoms from brain solitary metastasis without other metastases. They underwent surgery, radiotherapy and chemotherapy for solitary cerebral metastasis. Treatment of the brain lesion resulted in KPS improvement (KPS = 90) and survival was 16 and 30 months, respectively. From the 2 cases presented and the review of the literature, it appears that a better outcome may be obtained by a combined treatment of metastases, including surgery, radiotherapy and chemotherapy.

Cross-linked telomere-protein complexes from chicken erythrocyte nuclei: isolation by a new procedure.

DNA-protein cross-linkages were produced in intact nuclei of chicken erythrocytes by the action of cis-diammine dichloroplatinum. The telomeric DNA-protein cross-linked complexes were then isolated by hybridization with a biotinylated oligonucleotide and selective binding on immobilized streptavidin. Two main nonhistone proteins were present in the purified complexes, migrating in SDS-gel electrophoresis with apparent molecular masses of 66 and 58 kDa, respectively. Although the identity of these two proteins is still unknown, it is significant that two proteins with similar electrophoretic behavior have been described as constituents of the human telomeric complexes. This procedure could also be applied to the isolation of DNA-protein cross-linked complexes containing any chosen DNA sequence.

Metastatic spinal cord compression. Clinical remarks.

BACKGROUND: Metastatic compression of the spinal cord is a frequent occurrence throughout the evolution of neoplastic disease. Possible clinical-diagnostic strategies and therapeutic management of this pathology are discussed in terms of survival and quality of life. METHODS: The study includes 59 patients (40 males and 19 females, with an average age of 48.4 years) with metastatic spinal compression treated surgically in our centre (in some cases with stabilization of the spinal segment involved). RESULTS: In 40 cases the localization of the primary tumor was known when the patient was admitted. The segment involved was the dorsal one in 41 cases. The most frequent type of tumor was pulmonary carcinoma in males and breast carcinoma in females. Average survival was 5.3 months. Treatment integrated by stabilization improved the quality of life in comparison to laminectomy alone. Survival was also influenced by the histological type and site of the primary tumor. CONCLUSIONS: Surgical treatment not only prolongs survival but, above-all, guarantees a satisfactory quality of life.

[Therapeutic considerations in solitary cerebral metastases from uterine carcinoma]. / Considerazioni terapeutiche sulle metastasi cerebrali solitarie da carcinoma uterino.

Brain metastases from endometrial carcinoma rarely involve the nervous system and are solitary in exceptional cases (< 1% of cases). Two cases of solitary cerebral metastasis from endometrial carcinoma are described. Two patients, submitted to the therapeutic protocol established for endometrial carcinoma, underwent surgery, radiotherapy and chemotherapy for solitary cerebral metastasis after at average interval of 18 months. Average survival was 46 months and death was due to progression of the systemic disease. An examination of our cases and those described in the literature has shown that, although these metastasis do not respond well to therapeutic treatment, a better outcome may be achieved by combined treatment consisting of surgery, radiotherapy and chemotherapy.

Solitary plasmacytoma of the calvarium: a review of clinical and prognostic features.

Primary craniocerebral plasmacytomas are uncommon they represent only 0.7% of all plasmacytomas. We report one case of solitary plasmacytoma of the skull and discuss the clinical features and prognosis of this tumor. There seems to be no difference in prognosis between plasmacytomas originating from the bone (osseous form) and those originating from the dura mater (non-osseous form). In these lesions, the risk of secondary multiple myeloma appears to be low.

Intraosseous schwannoma of the vault of the skull.

Intraosseous schwannoma is a rare benign tumor of the bone with characteristic radiological and histological features. The most common sites of this tumor are mandible, sacrum, und vertebral bodies. Two cases, one of which is the first diagnosed with MRI, of this tumor in the vault of the skull are presented.

Intracerebral schwannoma. Case report.

A case of intraparenchymal schwannoma is presented. A 61-year-old woman, with stigmata of von Recklinghausen's neurofibromatosis (NF-2), presented with a history of weakness of the right lower limb for 2 months. She was investigated by MR which showed a circular mass with a maximum diameter of 5 cm in the right parieto-occipital lobe. The tumor was removed in toto via a left parieto-occipital craniotomy. The patient was discharged two weeks after the operation and remains well now 2 years later. The clinical and neuroradiological findings of reported intraparenchymal schwannomas, including the case reported here, are discussed.

[Epidermoid cyst of the cranial vault. Neuroradiological and therapeutic considerations]. / Cisti epidermoide della volta cranica. Considerazioni neuroradiologiche e terapeutiche.

AIM: Intradiploic epidermoid cyst is a slow-growing tumor affecting only rarely the cranial bones. PATIENT: The authors describe a case of intradiploic epidermoid cyst of the cranial vault in which there was a predominantly intracranial extension. Roentgenographic and CT findings do not permit a differential diagnosis. Complete removal of the cyst and its capsule was accomplished, with complete recovery. CONCLUSION: Total removal oft the tumor and its capsule is associated with a very good long-term prognosis without recurrences.

Epithelioid schwannoma of the ulnar nerve. Some clinical observations.

The authors describe one case of purely epithelioid schwannoma of the ulnar nerve and discuss the therapeutic management. A 44 year-old man was referred to us for a fusiform, movable mass in the left ulnar nerve and was removed by a wide en bloc excision. Intraoperative nerve action potentials were performed both prior to and following excision of the lesion. Histologically, the tumor was composed of round or polygonal cells arranged in necrotic clusters and anastomosing cords. There were areas of spindle cells. The epithelioid cells were round with abundant cytoplasm. Mitoses were frequent. S100 protein immunoreactivity was present diffusely in tumor cells (both nuclear and cytoplasmic), whereas cytokeratin, NSE, and anti-melanoma reactions gave negative results. After a 13 months, the patient's neurological conditions are excellent and there are no signs of either recurrence or metastasis.

Role of the dimeric structure in Cu,Zn superoxide dismutase. pH-dependent, reversible denaturation of the monomeric enzyme from Escherichia coli.

To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated. Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein. Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66 degrees C at pH 7.8 and at 79.3 degrees C at pH 6.0. The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources. Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose holo form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes. Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea. These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the beta-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its active-site region.

Sarcoma metastatic to the brain: a series of 15 cases.

METHODS: We report on 15 patients surgically treated for intraparenchymal brain metastases from sarcoma, including six osteosarcomas, five leiomyosarcomas, two malignant fibrous histiocytomas, and two alveolar soft-part sarcomas (ASPS). RESULT: Median survival after craniotomy was 9.3 months. Patients with a preoperative Karnofsky performance score of > 70 survived for 12.8 versus 5.3 months for those with a Karnofsky performance score < 70 (p=0.03). Patients with evidence of only lung metastases at the time of surgery (nine cases) survived 8.6 months, which was similar to the 10.4-month survival for patients with disease limited to the brain (p=0.1). The two patients with alveolar soft-part sarcomas are alive at 15 and 20 months after surgery. CONCLUSION: We conclude that surgery is effective in treating selected patients with sarcoma metastatic to the brain and that patients with metastasis from ASPS may have a relatively good prognosis if they are surgically treated. The complete removal of all brain metastases and a Karnofsky performance score > 70 are associated with a favorable prognosis; the presence of concurrent lung metastases is not a contraindication to surgery.