The specificity of prolyl endopeptidase from Flavobacterium meningoseptum: mapping the S' subsites by positional scanning via acyl transfer

The S1'-S3' subsite specificity of prolyl endopeptidase from Flavobacterium meningoseptum was studied by acyl transfer to libraries of amino acid amides and peptides. Whereas the S1' and S3' subsites influence the specificity for the amino component by approximately one order of magnitude, the S2' subsite possesses a markedly higher specificity. Besides the high specificity for hydrophobic residues at P1'-P3', proline was efficiently bound by the S2' and S3' subsites of the enzyme. In contrast, no binding of P1' proline-containing peptides was observed. It could be demonstrated that the specificity of the S' subsite is not restricted to L-amino acids. Effective P'-S' interactions were also found for beta- and gamma-amino acids indicating that the enzyme does not form close contacts to the backbone of P1' and P2' amino acid residues.     

The Wiskott-Aldrich syndrome

In 1937, Wiskott described three brothers with congenital thrombocytopenia, bloody diarrhea, eczema, and recurrent ear infections. Seventeen years later, Aldrich showed X-linked (a gene carried on the X chromosome) inheritance. Subsequently, the characteristic immune defects of Wiskott-Aldrich syndrome (WAS) were reported, including lymphopenia, lymphocyte depletion in the thymus, T-dependent pericortical areas of lymph nodes, defective delayed type hypersensitivity, and failure to produce antibodies to polysaccharides and to a variety of bacterial, protein, and viral antigens. The consistent platelet abnormalities were explained by ineffective thrombocytopoiesis. The increased risk of autoimmune diseases and malignancies was recognized. In addition to the classic WAS phenotype, a milder form designated as hereditary X-linked thrombocytopenia (XLT) has been described. The genes for both WAS and XLT have been mapped to Xp11.22 and sequence analysis has identified mutations of the same gene in both phenotypes. The gene coding for the WAS protein (WASP) is composed of 12 exons containing 1,823 base pairs and encodes a 502-amino acid protein. WASP is expressed in the cytoplasm of all hematopoietic stem cell-derived lineages. Although the precise function of WASP is unknown, several unique binding domains have been identified, and WASP appears to play a critical role in signal transduction by interacting with SH3-containing molecules and in the regulation of the cytoskeletal reorganization. The identification of the WASP gene allows the diagnosis of WAS on a molecular basis, carrier detection, and prenatal diagnosis. Treatment is largely symptomatic and includes antibiotics, prophylactic intravenous immunoglobulin (i.v.IG) and splenectomy in selected cases to reduce hemorrhages. Stem cell transplantation corrects the defect and should be considered in younger patients.     

Cellular uptake and localization of liposomal-methylphosphonate oligodeoxynucleotides

Nuclease digestion and intracellular delivery are major factors limiting the potential use of oligodeoxynucleotides as antisense molecules. Structural analogues of phosphodiester oligodeoxynucleotides, such as phosphorothioates and methylphosphonates, are resistant to nuclease degradation and can still bind to their mRNA targets. However, their limited ability to escape from the endosomal/lysosomal compartments and reach the intracellular sites of action have dampened their potential clinical application. To circumvent this problem we have incorporated methylphosphonate oligodeoxynucleotides into liposomes. We found that the level of uptake of liposome-incorporated methylphosphonate oligodeoxynucleotides is time and concentration dependent. Maximal up take occurred at 8 h when 4-8 microM liposome-incorporated methylphosphonate oligodeoxynucleotides was added. Approximately 50% of liposome-incorporated methylphosphonate oligodeoxynucleotides were retained in cells after 24 h of incubation. Using fluorescent microscopy, intracellular fluorescence could be seen within 2.5 h of incubation. Diffused fluorescence was found throughout the cytoplasm, suggesting that the liposome-incorporated methylphosphonate oligodeoxynucleotides were not confined within the endosomal/lysosomal structures. We conclude that liposomes can effectively deliver methylphosphonate oligodeoxynucleotides to the cytoplasm, which is the major intracellular site of action for translational arrest.     

Transcutaneous lower blepharoplasty

Transcutaneous lower blepharoplasty as a standard procedure for cosmetic lower blepharoplasty is being replaced by transconjunctival blepharoplasty and laser resurfacing techniques. Nevertheless, there remain specific indications for the transcutaneous approach and its modifications. The accomplished facial plastic surgeon will be able to choose the appropriate technique based on the patient's concerns, the eyelid pathology, and his or her clinical experience.     

Preoperative functional magnetic resonance imaging (fMRI) of the motor system in patients with tumours in the parietal lobe

Intracranial lesions may compromise structures critical for motor performance, and mapping of the cortex, especially of the motor hand area, is important to reduce postoperative morbidity. We investigated nine patients with parietal lobe tumours and used functional MRI sensitized to changes in blood oxygenation to define the different motor areas, especially the primary sensorimotor cortex, in relation to the localization of the tumour. Activation was determined by pixel-by-pixel correlation of the signal intensity time course with a reference waveform equivalent to the stimulus protocol. All subjects showed significant activation of the primary sensorimotor cortex while performing a finger opposition task with the affected and unaffected side. In five patients the finger opposition task additionally activated the ipsilateral sensorimotor cortex and the supplementary motor area (SMA). Extension and flexion of the foot, additionally performed in two patients, also activated the sensorimotor cortex, in one case within the perifocal oedema of the tumour. Tumour localization near the central sulcus induced displacement of the sensorimotor cortex as compared to the unaffected side in all patients with a relevant mass effect. The results of our study demonstrate that functional MRI at 1.5 T with a clinically used tomograph can reproducibly localize critical brain regions in patients with intracranial lesions.     

A quantitative method for LDH, MDH and albumin levels in tears with ocular surface toxicity scored by Draize criteria in rabbit eyes

We have established a quantitative method for evaluation of ocular surface lesions on the basis of lactate dehydrogenase (LDH) activity, malate dehydrogenase (MDH) activity, and albumin levels in rabbit tears. Lesions were produced with solutions of 0.005-0.02% benzalkonium chloride (BAK), 0.01-0.03% chlorhexidine digluconate (CHX), and 0.01-0.03% polyhexamethylene biguanide hydrochloride (PHMB), all of which are widely used in topical ophthalmic preparations. Two drops of test solution were instilled 15 times into rabbit eyes at 5 minute intervals. Sixty minutes after 0.02% BAK instillation, tear LDH activity increased from 1,840 U/L (without instillation) to 26,100 U/L, and concomitantly tear albumin levels rose from 0.11 mg/mL (without instillation) to 9.48 mg/mL. Instillation of 0.03% CHX and 0.03% PHMB caused smaller increases in LDH and MDH activity and albumin tear levels. LDH activity and albumin levels in tears were significantly correlated with the degree of total ocular surface lesions in both cornea and conjunctiva as observed by slit lamp biomicroscopy quantified using a modified Draize score. Based on the results of this study, we believe that LDH activity and albumin level in tears can be used as objective indicators for the quantitative evaluation of ocular surface lesions on both cornea and conjunctiva following application of topical ophthalmic preparations containing cytotoxic preservatives in animals and man.     

Cartilage-sparing otoplasty: a review with long-term results

Prominent ears are the most frequent congenital deformity in the head and neck region. Anatomy of normal and prominent ears as well as the psychological aspects of prominent ears are reviewed. Two types of surgical technique are described with emphasis on the cartilage-sparing technique. A sound pre-operative analysis, focusing on all parts of the deformity, and surgical techniques which are gradually applied to these deformities should result in pleasing, permanent changes for the vast majority of patients. In our opinion, a combination of cartilage-sparing techniques augmented with cartilage-weakening procedures give predictable long-term results with a natural appearing ear and concomitant few, easily treated complications.