Stoichiometry and heterogeneity of the pro-region chain in tetrameric human cathepsin C.

The subunit structure and composition of mature human cathepsin C, an oligomeric cysteine proteinase, has been characterised in detail. The heavy chain, light chain and pro-region peptides are shown to be held together solely by non-covalent interactions, and to be present in equimolar ratio, suggesting an important structural role for the residual pro-region chain which is strongly bound to the enzyme. The mass of the light chain, as determined by mass spectrometry, combined with its N-terminal sequence, determines the position of cleavage from the heavy chain. Amino-acid sequencing has led to definition of the 13.5 kDa N-terminal part of the pro-region which remains in the mature enzyme, the C-terminal moiety of 10 kDa being cleaved out and lost from the pro-peptide on activation. The residual pro-region is heterogeneous, a proportion being intact and the remainder being cleaved at alternative positions 58 or 61, yielding two smaller peptides joined by disulphide bond. The proportion of cleaved form was found to vary with tissue and enzyme preparation but did not affect enzyme activity. The molecular masses of the constituent chains after deglycosylation lead to a protein mass of 158 kDa. All four potential glycosylation sites are glycosylated.

Role of imprint cytology in the intraoperative evaluation of sentinel lymph nodes for malignant melanoma.

Controversy exists over the utility of different methods for intra-operative sentinel lymph node (SLN) evaluation in patients with malignant melanoma (MM). The aim of this study was to evaluate the role of intra-operative imprint cytology (IC) in patients with MM. 215 SLNs from 99 patients with MM were examined by IC and results compared with the results of permanent sections. 24 patients had MM deposits in their SLNs and this was confirmed by histological examination. Intraoperative IC was positive in 11 of these patients (46% sensitivity). In addition, there were three false-positive IC diagnoses (79% positive predictive value); one of these was due to contamination during the sectioning of the SLN. The specificity and the negative predictive values of the IC were 96 and 85%, respectively. IC is a valuable method of intra-operative SLN evaluation which can spare approximately half of the patients with clinically occult regional metastases from a second surgical procedure. However, special care must be taken to avoid false-positive results due to contamination.

Internal energy distributions deposited in doubly and singly charged tungsten hexacarbonyl ions generated by charge stripping, electron impact, and charge exchange.

The distribution Pε of internal energies deposited in W(CO)6 (+•). ions upon charge stripping (that is, electron detachment to yield the doubly charged ion in the course of a single kiloelec-tronvolt energy collision) was estimated by a thermochemical method from the measured relative abundances of the doubly charged fragment ions produced. The thermochemical information needed to estimate P/ge was obtained by measuring the threshold translational energy losses associated with charge stripping of the singly charged fragment ions, W(CO) n (+) (n = 0-5). The P(/ge) curve falls exponentially with increasing internal energy. The average energy transferred to W(CO)6 (+•) upon a 7.8-keV collision with O2 is 19 eV, yielding W(CO)6 (2•) ions with an average of 4 eV of internal energy. In its general appearance, the P(ε) distribution associated with charge stripping is similar to the curves obtained from simple collisional activation of either W(CO) 6 (+•). or W(CO)6 (2+•) in kiloelectronvolt energy gaseous collisions. Given that charge stripping occurs by way of an electronic excitation process, this similarity in the energy deposition function is taken to indicate that electronic excitation is also the major mechanism for simple collisional activation in this system at zero scattering angle in the kiloelectronvolt energy regime. The internal energy distribution associated with a related charge-stripping process, charge inversion from the metal carbonyl anions to yield the corresponding cations, was also recorded. This reaction shows a large (∼7 eV) average internal energy deposition with a distribution that indicates near-zero probability of formation of unexcited ions. These data are tentatively interpreted in terms of vibrationalelectron detachment. The internal energy distribution associated with an exothermic process, charge exchange [W(CO)6 (2+•) + O2 → W(CO) + (6•)+O2 (+•)], was also characterized. Unexpectedly strong coupling of translational to internal energy is observed, and there is a large probability of depositing internal energies in excess of 10 eV, even though the exothermicity is only 3 eV. Finally, the internal energy distributions associated with the formation of doubly charged W(CO)6 (2+•) ions by electron ionization have been measured. Unlike the distribution for charge stripping, but like that for singly charged ions generated by electron impact, this distribution shows considerable structure, presumably due to Franck-Condon factors.

Added value of blue dye in sentinel node biopsy for breast cancer.

Sentinel node biopsy in breast cancer is a new rapidly advancing minimal invasive procedure which enables nodal staging of clinically node negative breast cancer patients without performing complete axillary dissection. There are still controversies over the added value of Blue Dye when lymphoscintigraphy and gamma probe are used. In our series, 91 consecutive patients with invasive breast carcinoma were operated by a single surgeon, using lymphoscintigraphy, gamma probe and Blue Dye. The sentinel nodes (SLN) were histologically examined by HE and immunohistochemistry. Lymphoscintigraphy was succesful in 81 patients (89%). After the injection of Blue Dye, SLN could be identified in all 91 patients. Metastases in the SLN were present in 35 patients. We retrieved 128 SLN, of these 93 were hot and blue, 19 only hot and 16 only blue. The distribution of metastatic and nonmetastatic SLN between these three labeling groups was not different (P = 0.9361). We could not show any difference in the metastatic involvement of SLN in patients in whom preoperative lymphoscintigraphy could visualise the SLN preoperatively compared to those in whom it could not (P = 0.7315). False negativity calculated in our initial series of 36 patients was 0%. Our study showed added value of Blue Dye in detection of metastatic and nonmetastatic SLN.

Conservative treatment of female stress urinary incontinence with functional electrical stimulation.

BACKGROUND: Treatment of stress urinary incontinence with functional electrical stimulation (FES) is a recent and efficient method of conservative treatment. STUDY DESIGN: Before treatment, examinations determining the type of urinary incontinence should be made: patient history, pelvic and urologic examination, laboratory tests of urine, multi-channel urodynamic tests. The degree of urinary incontinence is assessed by pad tests. Only the patients with moderate stress urinary incontinence without or with a mild utero-vaginal prolapse, are treated by FES. The parameters of stimulation: the impulse is rectangular and biphasic, duration of impulse is 1 ms, frequency of impulse is 20 Hz, and intensity of current 35 mA. The chronic treatment with FES should be applied 1.5-2 h daily for 3 months. This long-term treatment requires appropriate motivation of the patient. RESULTS: Three months after terminated treatment 56 of the total 111 patients (50.5%) were cured, in 26 (23.4%) the condition improved, whereas in 29 patients (26.1%) the treatment failed. CONCLUSIONS: The efficiency of treatment depends on the patient selection, parameters of electrical stimulation, stimulator of the pelvic floor muscles, mode of stimulation--chronic stimulation, and on motivation of the patient.

Numerical investigation of fixed orthodontic brackets.

This paper presents the results of investigations into the mechanical behaviour of fixed orthodontic brackets. Special attention has been paid to the detailed modelling of the interface between the bracket base and an adhesive by which attachment is made to the tooth surface. Properties of this interface were taken as design parameters with the aim of investigating their influence on the stress field induced during bracket removal at the end of the treatment. Stress-strain analysis was performed using a finite element program developed in-house.

An evaluation of the quality of orthodontic attachment offered by single- and double-mesh bracket bases using the finite element method of stress analysis.

The objective of this study was to evaluate the influence of bracket base mesh geometry on the stresses generated in the bracket-cement-tooth continuum by a shear/peel load case. A validated three-dimensional finite element model of the bracket-cement-tooth system was constructed consisting of 15,324 nodes and 2971 finite elements. Cement geometric and physical properties were held constant and bracket base geometry was varied, representing a variety of single-mesh configurations and 1 double-mesh design. For the single-mesh designs, increasing wire diameter (100-400 microm) resulted in a decrease in enamel and cement stresses. Increases in wire mesh spacing (200-750 microm) increased the major principal stress recorded in the enamel and adhesive at all wire diameters. Within the bracket, the major principal stress increased significantly at wire spacing above 400-500 microm. However, within the impregnated wire mesh (IWM), the major principal stress decreased as wire space increased. When the double-mesh bracket base was considered, the combined mesh layers resulted in a decrease in the stresses recorded in the most superficial (coarse) mesh layer and an increase in the stresses recorded in the deepest (fine mesh) layer when compared with the single-layer designs in isolation. Modification of single-mesh spacing and wire diameter influences the magnitude and distribution of stresses within the bracket-cement-tooth continuum. The use of a double-mesh design results in a reduction in the stresses recorded in the most superficial mesh. Mesh design influenced stress distribution in this study, primarily by determining the flexibility of the bracket base.

The reliability and accuracy of intraoperative imprint cytology of sentinel lymph nodes in breast cancer.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is a new component of the surgical treatment of breast cancer that accurately predicts axillary status. In this study the authors evaluated the accuracy of intraoperative imprint cytology (IC) in comparison with definitive histologic evaluation of SLN in breast cancer patients. METHODS: A total 413 women with breast carcinoma and clinically negative axillary nodes underwent breast surgery and SLN biopsy. Mapping of SLN involved injection of (99m)Technecium labelled human albumin nanocolloid particles and Patent Blue dye. At the Department of Pathology, SLNs were bisected along its major axis. Both halves were imprinted 2-4 times on the slides and immediate staining with Hemacolor (Merck Germany) was performed for intraoperative examination. Imprint node negative women underwent no further surgery, while node positive women proceeded to full axillary clearance. Histological analysis of the SLN involved serial sectioning of the whole node with H&E and immunostaining for cytokeratin. RESULTS: Definitive histology revealed metastases (pN+) in 159/413 patients (38.5%): 69 (16.7%) macro metastases, 57 (13.8%) micro metastases, and 33 (8%) women with only isolated IHC positive cells or positive cell groups smaller than 0.2 mm (pNO sn+). The other 254 women had negative SLN biopsy. Imprint cytology detected 54/69 macro metastases, and 4/57 micro metastases. In the group with negative SLN (254), 2 cases were ''false positives''. CONCLUSIONS: Imprint of SLN biopsy can identify a negative axilla with high accuracy (specificity 99.2%). Overall sensitivity is only 36.5%, but macrometastases are detected in 77% which is important for performing ALDN in one session with operation of primary tumour.

[Indications for cerclage of the cervix uteri (author's transl)]. / Indikacije za serklazu.

Diagnostic methods for the incompetence of the internal cervical os are described. The incompetence of the internal cervical os when there is no pregnancy is especially dealt with. For the diagnosis five tests are used (measurement of the uterine cavity and palpation of the cervical channel, Palmer test, Bergman-Svennerund test cervicohysterography, and X-ray cervicometry). Only in strictly indicated cases should cerclage be applied. In cases with an undeveloped clinical picture the operation could be recommended twelfth week of pregnancy. With an appropriate selection of patients and an operation after the twelfth week of pregnancy a success of 82% was achieved at the university hospital in Ljubljana. The duration of the sick-leave after the operation is also indicated.

[Selection of patients in the treatment of urinary incontinence]. / Selekcija bolesnica u lijecenju urinske inkontinencije.

Criteria for the selection of female patients with urinary incontinence for treatment are described. The following procedures are suggested: the patient's history, gynecological examination, urological examination, urodynamic examinations (urethral pressure profile and cystometry), and neurophysiological examinations (especially the electromyography of pelvic floor muscles). For the treatment with functional electrical stimulation the role of the trial application of stimulators is emphasized. The importance of an accurate classification of incontinence is stressed. The results of treatment depend mainly on the appropriate selection of patients.

Effect of intra-abdominal position of the bladder neck and stability of its supporting structures on pressure transmission ratio after colposuspension.

The aim of this prospective study was to evaluate the influence of the higher intra-abdominal position of the bladder neck and the stability of its supporting structures after colposuspension, on pressure transmission to the urethra at the level of the bladder neck. Twenty-eight patients were included in the study. The pressure transmission ratio (PTR) was calculated at the level of the bladder neck, whereas the position and mobility of the bladder neck during coughing were evaluated with perineal ultrasound examination. The measurements were performed before and 3 months after colposuspension. After colposuspension we found a significant elevation of the PTR (P=0.001), a significantly higher intra-abdominal position (P=0.001) and decreased mobility (P=0.001) of the bladder neck during coughing. Also, a negative correlation between the elevation of PTR and decreased mobility of the bladder neck during coughing (r = -0.5049; P = 0.006) and a weak correlation between the elevation of PTR and a higher intra-abdominal position of the bladder neck during coughing were found (r = 0.3828; P=0.044). Reinforced tension resistance of the bladder neck supporting structures seems to be more important than intra-abdominal position of the bladder neck in achieving effective pressure transmission after colposuspension.

Selection of patients with urinary incontinence for application of functional electrical stimulation.

Contradictory data from the literature along with our own experience using functional electrical stimulation (FES) have facilitated the determination of objective criteria for the application of FES in correcting urinary incontinence. Simultaneous urodynamic, neurophysiological, and radiological examinations employed during our studies enabled us to determine changes in these parameters due to FEX. Among a large number of patients with urinary incontinence (for example: paraplegics, patients with spina bifida, stress incontinence following prostate operations, or resistant enuresis) we have determined that FES is appropriate if applied to properly selected patients. FES is indicated on the basis of the following criteria: the degree of morphological lesion of the urinary tract, the peripheral denervation of the muscles of the pelvic floor, the condition of the spinal center for miction, and, above all the quality of response to FES. FES not only activates the closing muscles of the bladder, but also inhibits the reflex contractions of the detrusor. Here, it should be noted that FES is indicated for cases of reflex incontinence. During FES of the pelvic floor muscles intravesical pressure normally diminishes and the reflex contractions of the detrusor subside. Sometimes, however, the reflex of miction occurs. In such a case, two reflex mechanisms are engaged. Their equilibrium, i.e. controlled miction, can be achieved with an afferent in flow which inhibits the reflex of miction. The use of FES for the correction of urinary incontinence can be a very good method if the FES parameters are individually chosen and patients properly selected.

The influence of obesity, constitution and physical work on the phenomenon of urinary incontinence in women.

Urinary incontinence in women is a common and complex problem which can be defined and classified as stress, urge and mixed incontinence. Three of the eight most common risk factors are obesity, constitution and physical work, in addition to age, length of menstrual cycle, number of pregnancies, education and level of health awareness. Women with the diagnosis of urinary incontinence were invited to respond to questionnaires on a voluntary basis. The three factors found to be associated with urinary incontinence are increased body weight, strong osteomuscular structure and hard physical work. These indicate that the work of a health team must take a holistic approach to women even before the phenomenon of urinary incontinence occurs.

The mechanisms of the action of electrical stimulation of muscles.

The mechanisms of the action of electrical stimulation of muscles are discussed. We believe that the principal cause for the good results obtained in cases of stress incontinence with electrical stimulation must be due to the formation of a better reflex in contraction of the muscles of the pelvic floor. This, however, envolves changes at the level of the spinal cord. By the help of stimulation the patient learns to use the urethral sphincter and secondary forces of retention in a better and a more proper way. The good results obtained in the treatment of some cases of nocturnal enuresis by anal plugs require yet another explanation. It can be considered that in nocturnal enuresis the unconscious inhibition of the reflex mechanism for emptying the bladder is not developed enough. From the experience we know that vigorous voluntary activation of the muscles of the pelvic floor inhibits the contraction of the detrusor muscle. In this way it is possible to depress the urging sensation to urinate. In the paper we have tried to demonstrate this mechanism objectively. On the basis of our findings we believe that the good results in treating nocturnal enuresis may be due to this mechanism of stimulation.

Reactivity of anomeric 1alpha- and 1beta-pentofuranosyl azide derivatives in ammonia chemical ionization.

Electron impact (EI), fast atom bombardment (FAB) and ammonia chemical ionization [CI(NH3)] mass spectrometry were applied with the aim of differentiating between the anomeric 1alpha- and 1beta-azidopentofuranosyl derivatives. Calculated ammonium affinities [AA(M)] and proton affinities [PA(M)] show that beta-anomers have higher affinities for H+ and NH4+ ions than alpha-azides. Protonated molecules, obtained by CI(NH3) of azidofuranosyl derivatives, lose HN3 giving abundant furanosyl (S+) ions. Ammonia solvation of MH+ ions competes with the previous reaction producing the [SNHN2NH3]+ ion, a competitive product to the ammonium-attached [SN3NH4]+ ion. The fragmentation pathways of the stable and metastable [MNH4]+, MH+ ions, and several other important fragment ions, were determined using mass analyzed ion kinetic energy spectrometry (MIKES). The abundance of the [SN3NH4]+ and/or [SNHN2NH3]+ ions was found to correlate inversely with the exothermicity of ammonia solvation of the MH+ ion. The abundance of the fragment ions [SNHNH3]+, [SNH3]+ and SNH+ in some examples correlates with the exothermicity of the corresponding [MNH4]+ and MH+ parent ion formation. The fragment ions SNH3+ and SNHNH3+ can be formed, at least in part, in the ammonia solvation reaction of the S+ and SNH+ ions taking place within the high-pressure region of the CI ion source.

Advances in nonimplantable electrical stimualtors for correction of urinary incontinence.

The nonimplantable electrical stimulators are widely used as rehabilitation aids for correction of urinary incontinence. The advances in the field of the design of nonimplantable electrical stimulators such as automatic vaginal electrical stimulator VAGICON-X and anal pressure controlled electrical stimulator are described. The evaluation of VAGICON-X in patients suffering from stress and urge incontinence as well as preliminary results of acute application of anal pressure electrical stimulation in patients with stress incontinence as presented.

Determination of submicrogram quantities of cimetidine and its biotransformation and degradation products.

The new and convenient method for the separation, isolation and characterization of the N-ciano-N'-methyl-N"-(2-/(5-methylimidazol-4-yl)methylthio/ethyl) guanidine and its biotransformation and degradation products was developed. Mass spectra obtained with the FAB (Fast Atom Bombardment) method are described. This method proved to be very successful for the analysis and characterization of studied compounds.

Bioconversion of steroid glycosides by Nocardia restricta.

The bioconversion of steroid alkaloid tomatine by Nocardia restricta yields the conjugate with lactic acid. We studied the bioconversion of some steroid glycosides without a nitrogen atom in the molecule to determine the effect of the nitrogen atom. The glycosides were of three different types: sterol glycosides, bufadienolide rhamnoside and steroid saponine. The results of bioconversions showed that Nocardia restricta converts steroid glycosides differently according to the sugar bound to the steroid aglycone. It can be concluded that in the absence of a nitrogen atom in the steroid molecule no conjugation with lactic acid by Nocardia restricta occurs.