Contribution of routine cardiac biological markers to the etiological workup of ischemic stroke.

BACKGROUND: Optimization of the detection of atrial fibrillation following stroke is mandatory. Unfortunately, access to long-term cardiac monitoring is limited in many centers. The aim of this study was to assess the potential usefulness of three routine biological markers, troponin, D-dimers and BNP, measured in acute stroke phase in the selection of patients at risk of cardio-embolic stroke. METHODS: Troponin, D-Dimers and BNP were measured within 48 h after admission for ischemic stroke in 634 patients. Stroke mechanism was defined at the 3 months follow-up visit using ASCOD classification using a standardized work-up. Association between clinical, radiological and biological markers and stroke mechanism was evaluated using logistic regression analyses. RESULTS: 159 patients (25.1% of total study population) had a cardiac mechanism. On multivariate analysis, admission initial stroke severity (OR 1.04, 95 CI% 1.004-1.07, p < 0.05) history of heart failure (OR 3.03, 95% CI 1.19-7.73, p < 0.05), ECG abnormalities and high BNP value (OR 4.34, 95% CI 2.59-7.29, p < 0.05) were associated with pure cardiac stroke mechanism. CONCLUSION: High BNP value measured within 48 h after stroke admission is an independent predictor of cardiac stroke mechanism. Its measurement might be used to improve the selection of patients for whom further cardiologic investigations such as continuous long term ECG monitoring would be the most useful. BNP should be added to the standard admission-work-up for stroke patients.

Molecular evidence supporting the neoplastic nature of odontogenic keratocyst: a laser capture microdissection study of 15 cases.

AIMS: The bland histology of odontogenic keratocyst (OKC) belies its capacity for aggressive behaviour. Genetic alterations of OKC have not been well studied. We examined the frequency and pattern of allelic imbalance on five different chromosome regions from 15 patients with OKC. METHODS AND RESULTS: Laser-assisted microdissection was performed on formalin-fixed paraffin-embedded tissue. Polymerase chain reaction analysis of extracted DNA targeted five polymorphic DNA markers (D3S1285, D9S161, D11S1316, D13S290, and TP53) representing chromosome regions 3p14, 9p21, 11q23, 13q12.1 and 17p13, respectively. All 15 cases of OKC were informative at a minimum of three of five loci, with 11 informative on all five loci. Twelve of 15 cases (80%) demonstrated loss of heterozygosity (LOH). Seven cases (47%) showed LOH at more than two DNA loci. The frequency of LOH was 5/11 (45%) at D3S1285, 3/15 (20%) at D9S161, 4/14 (29%) at D11S1316, 8/14 (57%) at D13S290 and 3/15 (20%) at TP53. CONCLUSIONS: The majority of OKCs harbour chromosomal abnormalities. This finding supports the supposition that OKCs are neoplastic. Furthermore, OKCs harbour allelic loss at some of the same loci identified in squamous cell carcinoma. This may aid in explaining the rare occurrence of squamous cell carcinoma arising in OKC.

Homology modeling and molecular interaction field studies of alpha-glucosidases as a guide to structure-based design of novel proposed anti-HIV inhibitors.

For AIDS therapy, there are currently a number of compounds available for multiple targets already approved by the FDA and in clinic, e.g. protease inhibitors, reverse transcriptase inhibitors (NRTI, NNRTI), fusion inhibitors, CCR4, CCR5 among others. Some pharmaceuticals act against the virus before the entrance of HIV into the host cells. One of these targets is the glucosidase protein. This novel fusion target has been recently explored because the synthesis of viral glycoproteins depends on the activity of enzymes, such as glucosidase and transferase, for the elaboration of the polysaccharides. In this work we have built an homology model of Saccharomyces cerevisiae glucosidase and superimposed all relevant glucosidase-like enzymes in complex with carbohydrates, and calculated as well molecular interaction fields in our S. cerevisiae active site model. Our results suggest that there are two saccharide binding sites which are the most important for the binding of inhibitors with this family of enzymes which supports the possibility of inhibitors containing only two sugar units. Based on these results, we have proposed a novel pseudo-dissacharide which is a potential pharmaceutical for AIDS treatment.

Condyloma acuminatum and condyloma-like lesions of the oral cavity: a study of 11 cases with an intraductal component.

AIMS: We studied the clinicopathological features of 11 condyloma and condyloma-like lesions of the oral cavity with an unusual mixed pattern of exophytic and intraductal growth. The latter manifest as involvement of minor salivary gland ducts by the proliferative squamous lesions. This pattern of ductal involvement has not been previously described in oral condyloma. METHODS AND RESULTS: The clinical history was available for nine patients ranging in age from 17 to 73 years. Two were female and seven male. The buccal mucosa (five cases) was the most common site of occurrence, followed by the floor of mouth (two cases), lingual frenum (two cases), and hard palate (one case). All lesions exhibited exophytic and intraductal growth. The latter manifested itself as extension of the lesions into the excretory ducts of minor salivary glands. Underlying minor salivary glands, present in many of the excisional biopsy specimens, typically showed changes of obstructive atrophy. The exophytic components of all cases exhibited some degree of parakeratosis, and cryptic invaginations of parakeratin were typically present. Koilocytes were present in seven lesions and were equivocal in four. Mucous cells were present in the intraductal component of all cases and the intraductal component was never keratinized, but often papillary. A mild stromal-based, lymphocytic host response was present in three. A variably prominent neutrophilic infiltrate was present in the exophytic component of eight. Dysplasia was not present in any case. Five of 11 cases were positive with anti-human papillomavirus (HPV) and two of 11 cases were positive for in-situ hybridization probes directed against HPV 6/11. All cases were negative for HPV 16/18 and 13/33/35. CONCLUSIONS: Oral condyloma acuminatum may involve the excretory ducts of minor salivary glands. The diagnosis of oral condyloma acuminatum is difficult, as these lesions share considerable histological overlap with squamous papilloma. Finally, the relationship between these two lesions is incompletely understood.

Early soft tissue pathosis associated with impacted third molars without pericoronal radiolucency.

OBJECTIVES: This study was performed to histologically evaluate soft tissue pathosis in pericoronal tissues of impacted third molars that did not exhibit pathologic pericoronal radiolucency. STUDY DESIGN: One hundred impacted third molars without evidence of abnormal pericoronal radiolucency (follicular space <2.0 mm) were removed for reasons other than participation in this study, and the pericoronal tissues were submitted for histopathologic examination. Specimens were fixed and processed routinely and were stained with hematoxylin and eosin before independent evaluation by each of 2 oral pathologists. A subset of both diseased and healthy tissues underwent additional evaluation for the presence of proliferating cell nuclear antigen (PCNA) for assessment of cellular activity. RESULTS: Of the specimens submitted, 34% showed squamous metaplasia suggestive of cystic change equivalent to that found in dentigerous cysts. Soft tissue pathosis was significantly higher in patients over 21 years of age (P =.001). Five of 8 diseased specimens demonstrated PCNA uptake, whereas none of 10 healthy specimens were PCNA positive. CONCLUSIONS: These findings suggest that radiographic appearance may not be a reliable indicator of the absence of disease within a dental follicle. We conclude that the incidence of soft tissue pathologic conditions is higher than generally assumed from radiographic examination alone.

Benign mixed odontogenic tumors.

As a group, the mixed odontogenic tumors histologically resemble various stages of tooth formation (odontogenesis). Because of this, confusion arises in diagnosis and nomenclature unless one is familiar with normal tooth development and its subsequent resemblance to the neoplasms and hamartomas which arise from the tooth-forming tissues of the jaws. This article reviews odontogenesis and relates it to the formation of the mixed odontogenic tumors-the ameloblastic fibroma, ameloblastic fibro-odontoma, and the odontomas. Correlation of clinical and radiographic features with the histologic features will generally result in correct diagnosis and proper treatment.

Linear IgA disease histopathologically and clinically masquerading as lichen planus.

In each of 2 cases reported, the patient presented with features of erosive lichen planus or lichenoid drug eruptions and an incisional biopsy taken from the patient was diagnosed histologically as lichen planus. Subsequent recurrences or exacerbations were associated with vesiculobullous lesions. Simultaneous or subsequent direct immunofluorescence studies--from the same tissue sample in one case and from a similar site in the other case--demonstrated classic features of linear IgA disease. Both patients were originally treated for lichen planus with systemic and/or topical corticosteroids with limited success. One patient was treated with sulfapyridine with minimal improvement. Both patients were subsequently treated with dapsone and demonstrated significant clinical improvement. We propose that linear IgA disease may be more common than reported in the oral cavity, inasmuch as many cases of recalcitrant lichen planus, erosive lichen planus, and lichenoid drug eruptions, especially those with a vesiculobullous component, may in reality represent linear IgA disease. We recommend that direct immunofluorescence be done in any case in which bullous lichen planus is suspected.

Intraosseous fibrous lesions of the jaws: a manifestation of tuberous sclerosis.

Four patients previously diagnosed with tuberous sclerosis are reported with intraosseous fibrous lesions of the jaws. Review of the literature revealed comparable pathosis occurring in extragnathic bones and several previous reports of similar lesions within the jaws. Therefore, these intraosseous fibrous proliferations are thought to represent an intraoral manifestation of tuberous sclerosis and not coincidental findings. In all 4 cases, the tumors demonstrated significant collagenization with numerous interspersed plump fibroblasts. Although histopathologically similar, the features of the lesions are not specific and also can be found in desmoplastic fibromas and simple odontogenic fibromas. The definitive diagnosis requires appropriate clinicopathologic correlation.

Cation binding to the integrin CD11b I domain and activation model assessment.

BACKGROUND: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The alpha chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11 b I domain and to evaluate the structural effects of divalent ion binding to this protein. RESULTS: We have determined the X-ray structure of a new crystal form of the CD11 b I domain in the absence of added metal ions by multiple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straight-forward assessment of the structural effects of divalent cation binding at the metal ion dependent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein conformation and metal-ion coordination of the I domain is the same as that observed for all previously reported CD11 a I-domain structures and a CD11 b I-domain complex with Mn2+. These structures define a majority conformation. CONCLUSIONS: Addition of the cations Mg2+, Mn2+ and Cd2+ to the metal-free I domain does not induce conformational changes in the crystalline environment. Moreover, we find that Ca2+ binds poorly to the I domain which serves to explain its failure to support adhesion. We show that the active conformation proposed by Lee et al, is likely to be a construct artifact and we propose that the currently available data do not support a dramatic structural transition for the I domain during counter-receptor binding.

Recombinant human cytomegalovirus protease with a C-terminal (His)6 extension: purification, autocatalytic release of the mature enzyme, and biochemical characterization.

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli. The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E. coli as inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E. coli. In addition, the refolded CMV PR could be crystallized for X-ray diffraction.

Desmoplastic variant of ameloblastoma in an 83-year-ald Asian female: report of a case with literature review.

The desmoplastic ameloblastoma is a rare histologic variant of ameloblastoma. This presentation outlines the successful treatment of an 83-year-old Asian female with this type of tumour by surgical resection and peripheral ostectomy with preservation of the interior border of the mandible. Clinical, radiographic, histopathologic and surgical aspects of this tumour were reviewed.

Oral mucosal melanomas: the WESTOP Banff workshop proceedings. Western Society of Teachers of Oral Pathology.

A workshop to discuss primary oral melanomas was convened at the annual Western Society of Teachers of Oral Pathology meeting in Bannf, Alberta, Canada. Fifty oral melanomas, identified from the files of the participants, were reviewed in order to better understand the clinical features, histologic spectrum, and natural history of these perplexing lesions. Results confirmed that oral melanomas occur in adults almost three times more frequently in men than women and have a decided predilection for the palate and gingiva. Some lesions exhibit a clinically detectable and prolonged in situ growth phase, whereas others seem to lack this property and exhibit only or predominantly invasive characteristics. Recurrences, metastases, and death from tumor were characteristic of the follow-up of a limited number of patients. Until definitive prospective data are collected that elucidate natural history, oral mucosal melanomas should be tracked separately from cutaneous lesions. All oral pigmented lesions that are not clinically diagnostic should be biopsied. Lesions with equivocal histopathologic features might be referred to as "atypical melanocytic proliferation" and should be excised. Recognition of lesions in an early in situ phase and aggressive treatment should have a favorable effect on prognosis. To enhance future or prospective study of these rare neoplasms, guidelines for reporting oral melanomas are suggested.

Fluorescence and site-directed mutagenesis studies of interleukin 1 beta.

The authors mutated two key residues in the sequence of the cytokine interleukin 1 beta, namely the double mutant Phe46 to Trp46 and Trp120 to Phe120 and the single point mutation Lys103 to Leu103 and measured the resulting receptor binding and biological activities. The biological and receptor binding activities of the Trp46 mutein was reduced by a factor of 12 and 25, respectively, and surprisingly, those of the Leu103 mutein, 2600 and 600-fold relative to the wild-type protein. The authors had previously showed that Lys103 was unusually reactive to a variety of derivatizing agents. Furthermore, the Trp to Phe mutation allowed us to monitor the local environment of that residue by studying its intrinsic fluorescence properties, as well as any change in the fluorescence properties of Trp120 of the Leu103 mutein. The results of these studies show that mutation of Lys103 to Leu103 produces subtle long-range changes in the micro-environment of Trp120, indicative of a key role for this residue in the folding of the entire protein.

Structure-based design of novel HIV protease inhibitors: carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent nonpeptidic inhibitors.

The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover nonpeptidic HIV protease inhibitors had previously identified compound II (phenprocoumon, K(i) = 1 muM) as a lead template. Crystal structures of HIV protease complexes containing the peptide-derived inhibitor I (1-(naphthoxyacetyl)-L-histidyl-5(S)-amino-6-cyclohexyl-3 (R),4(R)-dihydroxy-2(R)-isopropylhexanoyl-L-isoleucine N-(2-pyridylmethyl)amide) and nonpeptidic inhibitors, such as phenprocoumon (compound II), provided a rational basis for the structure-based design of more active analogues. This investigation reports on the important finding of a carboxamide functionally appropriately added to the 4-hydroxycoumarin and the 4-hydroxy-2-pyrone templates which resulted in a new promising series of nonpeptidic HIV protease inhibitors with improved enzyme-binding affinity. The most active diastereomer of the carboxamide-containing compound XXIV inhibited HIV-1 protease with a K(i) value of 0.0014 muM. This research provides a new design direction for the discovery of more potent HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.

Focal cemento-osseous dysplasia: a clinicopathologic study of 221 cases.

Classification of cemento-osseous lesions of the jaws has long been a dilemma for pathologists. A group of 221 cemento-osseous lesions exhibited sufficiently distinctive clinicopathologic features to be separated into a specific category: focal cemento-osseous dysplasia. This entity presents as an asymptomatic, focal, mixed radiolucent/radiopaque lesion with ill-defined borders in the tooth-bearing areas. It was found to occur with greater frequency in women (88%) and in the posterior mandible (77%). The average age at presentation was 37 years and a relative predilection for black patients was observed. At surgery these lesions were noted to be hemorrhagic, gritty, and adherent to the surrounding bone. The gross appearance of multiple hemorrhagic fragments is of diagnostic significance. Histologic features include a cellular connective tissue stroma punctuated by irregular osseous and/or cementum-like calcifications. Focal cemento-osseous dysplasia is thought to be of periodontal ligament origin and to be non-neoplastic in nature. Further surgical intervention is not necessary, but periodic follow-up is recommended, because occasional cases were observed to progress into florid osseous dysplasia. Care must be taken to differentiate focal cemento-osseous dysplasia from central cementifying and/or ossifying fibromas, which are true neoplasms and require surgical treatment.

The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties.

Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study. Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2'. Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, however, were not viable enzymatically because of poor physical characteristics. This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme. Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease. Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.

Isolation and analysis of novel mutants of Escherichia coli prlA (secY).

Plasmid libraries of prlA mutants containing single-base-pair changes throughout the gene were generated by in vitro random mutagenesis. The prlA mutations capable of suppressing the secretion defect of LamB caused by mutations in the LamB signal peptide were selected and analyzed. Together with additional mutations generated by site-directed mutagenesis, a number of novel prlA mutations and/or suppressors were identified. These mutations provide the starting points for studying the relationship of structure and function of PrlA in its interaction with LamB and/or other component(s) in the Escherichia coli protein secretion-translocation complex.