The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1.

BACKGROUND: Granzyme B, one of the most abundant granzymes in cytotoxic T-lymphocyte (CTL) granules, and members of the caspase (cysteine aspartyl proteinases) family have a unique cleavage specificity for aspartic acid in P1 and play critical roles in the biochemical events that culminate in cell death. RESULTS: We have determined the three-dimensional structure of the complex of the human granzyme B with a potent tetrapeptide aldehyde inhibitor. The Asp-specific S1 subsite of human granzyme B is significantly larger and less charged than the corresponding Asp-specific site in the apoptosis-promoting caspases, and also larger than the corresponding subsite in rat granzyme B. CONCLUSIONS: The above differences account for the variation in substrate specificity among granzyme B, other serine proteases and the caspases, and enable the design of specific inhibitors that can probe the physiological functions of these proteins and the disease states with which they are associated.

Phosphinic acid inhibitors of D-alanyl-D-alanine ligase.

We report the synthesis of a series of phosphinic acid dipeptide analogues, NH2CH(R1)PO(OH)CH2CH(R2)CO2H, related to DAla-DAla. The best of these compounds are potent, essentially irreversible inhibitors of DAla-DAla ligase, and their preferred stereochemistry was shown by chiral synthesis of (1(S)-aminoethyl)(2(R)-carboxy-1-n-propyl)phosphinic acid, 12b, and by X-ray crystallography of its derivative benzyl [1(S)-[(benzyloxycarbonyl)-amino]ethyl](2(R)-carbomethoxy-1-propyl) phosphinate, 13, to correspond to the stereochemical configuration of DAla-DAla at both centers. A mechanism for the inhibition of DAla-DAla ligase by these compounds is proposed to involve an ATP-dependent formation of phosphorylated inhibitor within the enzyme's active site. The antibacterial activities of these compounds are modest although their spectra include both Gram-positive and Gram-negative susceptible organisms. The best antibacterial activity was shown by (1(S)-aminoethyl) [2-carboxy-2(R)-(methylthio)-1-ethyl]phosphinic acid, 3e, whose MIC's range from 4-128 micrograms/mL on nine of a panel of 11 bacterial organisms. Combination of one of the more active phosphinic acids 12b with the alanine racemase inhibitor fluoro-D-alanine enhances the antibacterial spectrum of the latter on several strains of bacteria and inhibits fluoro-D-alanine's self-reversal, which normally occurs at concentrations several fold higher than its MIC level. This inhibition of fluoro-D-alanine self-reversal is consistent with an involvement of DAla-DAla ligase inhibition in the antibacterial activity of these compounds.

Inhibition of rat alpha-reductases by finasteride: evidence for isozyme differences in the mechanism of inhibition.

The mechanism of inhibition of the rat types 1 and 2 5alpha-reductase by finasteride was investigated using recombinantly expressed enzymes. These studies revealed that finasteride is a potent, reversible inhibitor of the rat type 1 5alpha-reductase with Ki=10.2+/-1.3 nM. Finasteride is a potent inhibitor of the rat type 2; however, in this case the compound binds to the type 2 isozyme-NADPH complex to form a ternary complex with Ki=1.19+/-0.10 nM, which then rearranges to a high affinity complex (E:I) with a pseudo first order rate constant of 1.62+/-0.22 x 10(-3)/s. The second order rate constant is k3/Ki=1.37+/-0.31 x 10(6) M/s. Heat denaturation of the (type 2 enzyme:inhibitor) complex releases dihydrofinasteride and presumably the NADP+-adduct previously identified with the human 5alpha-reductases. The effects of finasteride were also studied in intact COS cells transiently expressing the rat types 1 and 2 5alpha-reductase. Results with whole cell assays confirm differences in mechanism of inhibition of rat types 1 and 2 5alpha-reductase by finasteride.

Cloning, expression and characterization of rhesus macaque types 1 and 2 5alpha-reductase: evidence for mechanism-based inhibition by finasteride.

The rhesus macaque types 1 and 2 5alpha-reductase (5aR1 and 5aR2) were cloned and expressed in COS cells to facilitate comparison of rhesus and human 5aRs. The deduced protein sequences of the rhesus SaRs shared 94% and 96% identity with the human type 1 and 2 isozymes, respectively. Despite a four amino acid insertion at the N-terminal region of rhesus 5aR1, the biochemical properties of rhesus and human homologs are very similar with respect to pH optimum, Km values for testosterone and progesterone, and inhibition by a variety of inhibitors. As expected, the biochemical properties of the human and rhesus 5aR2 are also very similar. The mechanism of inhibition of the rhesus 5aR1 and 5aR2 by finasteride was investigated in more detail. Finasteride displays time dependent inhibition of the rhesus 5aR1 and 5aR2 with second order rate constants of 4 x 10(3) M(-1) s(-1) and 5.2 x 10(5) M(-1)s(-1). Inhibition of rhesus 5aR2 with 3H-finasteride resulted in 3H bound to the enzyme which is not released by dialysis. Heat denaturation of the [rhesus SaR2:inhibitor] complex releases dihydrofinasteride, a breakdown product presumably related to the NADP+-adduct previously identified with the human SaRs (Bull et al., Mechanism-based inhibition of human steroid 5alpha-reductase by finasteride: Enzyme catalyzed formation of NADP-dihydrofinasteride, a potent bisubstrate analog inhibitor. J. Amer. Chem. Soc., 1996, 118, 2359-2365). Taken together, these results provide good evidence that the rhesus macaque is a suitable model to evaluate the pharmacological properties of finasteride and other 5aR inhibitors.

[Current aspects of facelift surgery]. / Aktuelle Aspekte der Facelift-Chirurgie.

In the care of the aging face, the facelift procedure occupies the center of attention. Of the many techniques available, only a few procedures fulfill the justifiable expectations that both patient and physician should have of such an intervention to reach the four goals of a facelift operation: create a natural, nonoperated appearance, obtain long-term durability, ensure a minimal complication rate, and restore or maintain a youthful vibrancy. This can especially be achieved with the so-called super-extended face lift with SMAS dissection, rotation, and refixation. Besides possessing surgical skill, every surgeon working in the field of aesthetic surgery must have a "concept of beauty" as defined by Connell and Levy, i.e., the surgeon must recognize the entirety of the face as an aesthetic unit and plan each intervention on an individual basis. Thus, in many cases it is not only necessary to correct the cheek and neck area, but also to take the forehead/eyebrow section into consideration.

[Value of genioplasty as a profile improving intervention in esthetic facial surgery]. / Bedeutung der Genioplastik als profilverbessernder Eingriff in der ästhetischen Gesichtschirurgie.

Genioplasty with rigid fixation using miniplates, preservation of vascular supply and accurate refixation of the soft tissue results in accurately predictable and stable bone and soft tissue contours. The Krefeld study of 36 patients who underwent genioplasty showed a consistent ratio of 85:100 of soft tissue to bone by advancement genioplasty and a 81:100 ratio by set back genioplasty.

[Esthetic facial surgery]. / Asthetische Gesichtschirurgie.

In the last few years, aesthetic facial surgery--especially soft-tissue surgery--has received increasing attention, not only from the medical profession but also particularly from the media. This is due, in part, to the growing level of general acceptance of the formerly stigmatized issue of aesthetic surgery, and in part to increasing patient expectations. Moreover, the introduction of less invasive procedures with outstanding long-term results have brought about a considerable change in aesthetic surgery. A comprehensive account of face lifting, forehead lifting, brow lifting, blepharoplasty, cervical liposuction and adjuvant techniques of skin rejuvenation will be given and discussed.

[Differential diagnosis and treatment of cheilognathopalatoschises]. / Differenzialdiagnostik und Therapie der Lippen-Kiefer-Gaumen-Spalten.

Cleft lip, alveolous and palate is the second frequent malformation in Europe with an incidence of 1 : 500. Pertaining to ontogeny it must be differentiated between cleft lip and alveolous and cleft palate. Cleft lip and cleft lip and alveolous can occur unilateral, right or left, or bilateral. Cleft bony palate can also occur unilateral, right or left, or bilateral, but cleft velum only in the median plane. Diagnostic and treatment of cleft lip and palate call for interdisciplinary cooperation between gynecologist/obstetrician, cranio-maxillo-facial surgeon, pediatrician, otorhinolaryngologist, orthodontist and logopedist. The schedule of primary cleft surgery in Germany is marked by a more-stage concept, in which at the end of the second year of life cleft lip and palate except cleft alveolous should be closed up. Despite of most careful surgery patients with cleft lip and palate can show functional and aesthetic disturbances. The functional disorders can affected masticatory function, speech, hearing and nasal breathing. Aesthetics disorders can be concerned to skeletal or soft tissue deformities of lip and nose. Operative corrections of bone and soft tissue can rehabilitate these patients entirely from functional and aesthetic view.

[Animal experimental studies on bone regeneration following drilling of the bone]. / Tierexperimentelle Untersuchungen zur Frage der Knochenregeneration nach Bohrvorgängen im Knochen

Experiments in dogs showed that drilling at the surface of bone provokes damage which increases with the number of revolutions per minute. Polychrome sequence marking and histological tests demonstrated that filling up of bony defects started later with turbine-driven drills than with drills of 2,000 r.p.m. and has not even been completed after eight weeks. It is therefore recommended to abstain from using turbines in drilling at the surface of bone.

Histologic subclassification of the cystadenolymphoma of the parotid gland. Analysis of 275 cases.

Cystadenolymphomas (CAL) of the parotid gland are variable in their epithelial differentiation and the ratio of the epithelial tumor component to lymphoid stroma. Two hundred and seventy five cases of CAL from the files of the Salivary Glands Register of the Institute of Pathology, University of Hamburg (1965-1979) were analysed. Their pathogenesis from parenchyma included in regional lymph nodes is discussed. The following subclassification was established. 1. Depending on to the ratio of epithelial tumor component to lymphoid stroma, three subtypes were distinguished. Subtype 1, "typical CAL" with an epithelial tumor component of 50%, amounted to 77% of all cases of CAL studied. Oncocytic differentiation and focal metaplasia to goblet cells or squamous epithelium was also found. 13.5% of CAL were classified as subtype 2, "stroma-poor CAL" with an epithelial tumor component of 70 to 80%. The tumor structure was similar to that of an oncocytoma in places. Two per cent of the CAL were in subtype 3, "stroma-rich CAL" with an epithelial tumor component of only 20 to 30%. Subtype 3 was found solely in men. The average age at presentation (61 years) was slightly lower than that of all the cases studied (65 years). 2. In 7.5% of the cases large areas of squamous cell metaplasia and regressive changes was found within a CAL. These cases were classified as subtype 4 ("metaplastic CAL"). The average age was 67 years. The case histories showed that 20% of these metaplastic CAL had previously been irradiated. 3. Bilateral CAL was found in 7.5% of the cases. In 4% multifocal CAL occurred in the parotid gland unilaterally. Recurrences were observed in 2% of all CAL. 4. Carcinoma in CAL is rare (we found two cases in our own material). In 50% of all cases reported radiotherapy was mentioned in the case histories. 5. Malignant tumors coincident with CAL were recorded in 3% of the cases. 6. The lymphoid stroma showed reaction patterns similar to those of the regional lymph nodes. These included granulomatous changes (foreign body granuloma with cholesterol deposits, tuberculosis) and tumor metastases. In the neighborhood of oncocytic tumor epithelium focal accumulations plasma cells forming IgA and IgG were found. Metaplasia to squamous epithelium is believed to be caused by circulatory disturbances, irradiation, and other noxae. In the differential diagnosis of the stroma-poor subtype 2, oncocytoma and cystic sialadenoma must be excluded, and in the differential diagnosis of subtype 4 (the metaplastic CAL), sebaceous adenoma, mucepidermoid tumor, squamous cell carcinoma, lymphoepithelioma, and other non-tumorous lesions of the parotid gland (lymphoepithelial cysts, myoepithelial parotitis) must be ruled out. Our findings suggest that CAL develops from parenchyma included in parotid lymph nodes with the oncocytic ductal epithelium representing the neoplastic component.

[Augmentation of the extremely atrophied maxilla and mandible by autologous calvarial bone transplantation]. / Aufbau des extrem atrophierten Ober- und Unterkiefers durch autologe Kalottenknochentransplantate.

Rehabilitation in patients with severe alveolar ridge atrophy of the maxilla or mandible is problematic and can often only be achieved by long-term treatment. In most cases, autologous bone grafting with iliac crest bone has been used to augment severely atrophied upper jaws. In our experience, iliac bone grafts are less useful, since iliac bone appears to be of inferior quality; in elderly osteoporotic women, the bone is soft, indentable, and of poor osteogenic potency. In our department, we have been using only autologous calvarial bone grafts for augmentation of alveolar ridge atrophy since 1993. The bone is removed from the outer table of the skull only, trimmed to the alveolar ridge, und fixed with titanium lag scews. The skull defect created is covered with crushed bone or a titanium mesh to avoid aesthetic problems. Insertion of dental implants follows after a healing period of the bone grafts of 5-6 months. A total of 63 patients underwent calvarial split-graft augmentation; augmentation of the maxilla and mandible was carried out in 15 of these patients, of the maxilla only in eight, and of the mandible only in 40. The investigations 1 year later showed a resorption rate of approximately 10%. This is lower than when using iliac bone grafting. The resorption results were stable between 6 and 12 months after augmentation. Using dental implants (12 patients with 32 implants), the resorption rate was low and constant. We have never seen total loss of bone grafts or intracranial complications. All patients were pleased with the treatment. In our opinion, severe alveolar atrophy of the maxilla or mandible should be compensated for by augmentation with autologous calvarial bone grafts to obtain good long-term results.

Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography.

Lisinopril (N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline), a potent angiotensin-converting enzyme inhibitor, is an exceptionally selective affinity chromatography ligand for this enzyme. Affinity chromatography furnishes electrophoretically homogeneous enzyme directly from crude homogenates of rabbit lung tissue, a 1,000-fold purification; also, it affords a 100,000-fold enrichment of the more rare human plasma enzyme in a single step. The affinity of angiotensin-converting enzyme for the Sepharose-spacer-lisinopril matrix (Ki matrix = 1 X 10(-5) M) is weak compared to its affinity for free lisinopril (Ki = 1 X 10(-10) M). The capacity of the affinity column is described quantitatively as a function of Ki matrix, lisinopril, and enzyme concentrations. The recovery of bound enzyme is low in chromatography of crude tissue samples (10-40%), although it approaches a reversible process (70-100%) with pure enzyme. The holoenzyme is converted to Zn2+-free apoenzyme to effect removal of lisinopril. In this process, the rate constant for spontaneous dissociation of Zn2+ from free enzyme is 1 X 10(-2) s-1 (t 1/2 = 1 min), which places a lower limit of 3 X 10(-10) M on the dissociation constant of Zn2+ at neutral pH from angiotensin-converting enzyme. The exceptional selectivity of lisinopril as an affinity chromatography ligand for angiotensin-converting enzyme suggests it is among the most specific inhibitors designed for any enzyme.

[Indications and technic of transdental fixation]. / Zur Indikation und Technik der transdentalen Fixation

Transdental fixation, as closed implant, offers the most favourable preconditions of all dental procedures of implantation. Teeth where the root-crown relation is altered to the detriment of root length, which is held firm in bone, can with its help be made functional. Loosening and premature loss of teeth can be prevented. Further discussed is the indication for this procedure, especially in tooth fractures and luxations as well as the operative steps. Up to now transdental fixation has proved itself although the number of cases and the follow-up time are insufficient to allow final judgment.

[Significance of hemodynamic parameters of blood loss in orthognathic surgery]. / Bedeutung hämodynamischer Parameter für den Blutverlust in der Dysgnathiechirurgie.

INTRODUCTION: The hemodynamic parameters of 95 patients undergoing maxillary or bimaxillary orthognathic surgery in 1996 and 1997 at the Department of OMF Surgery/Plastic Surgery, Krefeld, Germany, were analyzed retrospectivly to study the effect of intraoperative blood loss. MATERIALS AND METHODS: The parameters included the blood loss volume, age, weight and sex of the patients, the mode of osteotomy and the operation time, the surgeon, the average blood pressure, the infusion volume, the anesthesiologist, the thrombocyte counts and their function, the activity of the coagulation factors II, V, VII, VIII, IX, X, XI, XII, XIII and von-Willebrand-factor, and the pathological coagulation factor counts of each patient, the rate of autologous blood donation and the rate of retransfusion. Statistical analysis was done using the Speraman-Rhotest. RESULTS: The average blood loss during maxillary osteotomy was 670 +/- 380 ml and during bimaxillary surgery 1120 +/- 510 ml. Men lost about 300 ml more than women. Operations of more than 3.5 h in length led to a blood loss of 1200 +/- 520 ml as opposed to 670 +/- 310 ml. The average blood loss among various surgeons was between 670 ml and 1180 ml of various anesthesiologists between 730 ml and 1200 ml, without statistical evidence. Some 17.9% of patients showed pathological thrombocytic function concerning medication with aspirin; 34.7% had pathological activities of coagulation factors, but only 2.1% with clinical significance. CONCLUSION: Mode of operation, maxillary or bimaxillary, und length of operation were the most significant factors of intraoperative blood loss. Patients with pathological coagulation had nearly the same rate of blood loss as patients with physiological coagulation. In most cases this was determined by restriction of aspirin. Analysis of the rate of autologous blood retransfusion showed a significant correlation to blood loss in bimaxillary surgery. Maxillary osteotomy led to a retransfusion of only 14.2% of autologous blood unit. This should be reviewed critically especially concerning costs.

Isolation of an angiotensin II-binding protein from liver.

A protein that specifically binds angiotensin II has been isolated in nearly homogeneous form by two independent approaches after solubilization from rabbit liver particles by treatment with digitonin. The protein purified by either of these methods resembles in size the single radioactive macromolecular component made by using disuccinimidyl suberate to crosslink radioiodinated angiotensin II with its receptor in the solubilized extract. In the first technique, angiotensin II as an affinity ligand specifically extracted the protein from a preparation that had been freed of angiotensin-degrading activity. In the second approach, the angiotensin II-protein complex was specifically precipitated by anti-angiotensin II antibodies and staphylococcal protein A-Sepharose. The protein could be eluted from the affinity column with angiotensin II or 4 M MgCl2. The angiotensin II-protein complex dissociated in the presence of sulfhydryl-containing reagents, and these could therefore be used to elute it from either the chemical or the immunoaffinity-based matrix. This effect of sulfhydryl-containing reagents and the paradoxical observation that the isolated protein after denaturation exhibited a slower electrophoretic mobility in its reduced form that in its unreduced form suggest that the binding configuration of this protein may be sensitive to reduction.

Late results following different methods of cleft lip repair.

This is a review of the cleft lip repair of 232 patients. Three groups were compared, one repaired according to the Hagedorn-Le Mesurier-Steffensen technique, another by the Tennison method and a third using the Pfeifer wave-line procedure. Muscular union was best following Pfeifer's wave-line procedure. Both the Hagedorn-Le Mesurier-Steffensen and the Tennison lips were often too long, while the Pfeifer lips were more often too short. Reconstruction of Cupid's bow was somewhat more harmonious following Tennison's method. There was a trend towards fewer disturbing scars with the Pfeifer's procedure. We feel that these findings support our 1970 decision to operate all cleft lips according to the wave-line procedure.

Adenovirus L4-100K assembly protein is a granzyme B substrate that potently inhibits granzyme B-mediated cell death.

Cytotoxic lymphocytes kill virus-infected target cells and play a critical role in host recovery from viral infections. Granzyme B (GrB) is a cytotoxic lymphocyte granule protease that plays a critical role in mediating cytotoxicity. In these studies, we demonstrate that the adenovirus assembly protein L4--100K (100K) is a GrB substrate that prevents cytotoxic lymphocyte granule-induced apoptosis in infected target cells by potently inhibiting GrB. This inhibition is absolutely dependent on Asp-48 in 100K, found within a classic GrB consensus motif. 100K is the first viral protein described that exclusively targets the GrB pathway. It represents a novel class of viral protease inhibitor, in which an essential, multifunctional viral protein, which is vulnerable to specific proteolysis by GrB, expresses inhibitory function against that protease.

Inhibition of rabbit lung angiotensin-converting enzyme by N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline.

Two novel peptide analogs, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and the corresponding L-lysyl-L-proline derivative, have been demonstrated to be potent competitive inhibitors of purified rabbit lung angiotensin-converting enzyme: Ki = 2 and 1 X 10(-10) M, respectively, at pH 7.5, 25 degrees C, and 0.3 M chloride ion. Second-order rate constants for addition of these inhibitors to enzyme under the same conditions are in the range 1-2 X 10(6) M-1 s-1; first-order rate constants for dissociation of the EI complexes are in the range 1-4 X 10(-4) s-1. The association rate constants are similar to those measured for D-3-mercapto-2-methylpropanoyl-L-proline, captopril, but the dissociation rate constants are severalfold slower and account for the higher affinity of these inhibitors for the enzyme. The dissociation constant for the EI complex containing N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline is pH-dependent, and reaches a minimum at approximately pH 6: Ki = 4 +/- 1 X 10(-11) M. The pH dependence is consistent either with a model for which the protonation state of the secondary nitrogen atom in the inhibitor determines binding affinity, or one for which ionizations on the enzyme alone influence affinity for these inhibitors. The affinity of this inhibitor for the zinc-free apoenzyme is 2 X 10(4) times less than for the zinc-free apoenzyme is 2 X 10(4) times less than that for the holoenzyme. If considered as a "collected product" inhibitor, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline appears to derive an additional factor of 375 M in its affinity for the enzyme compared to that of the two products of its hypothetical hydrolysis, a consequence of favorable entropy effects.

Molecular and catalytic properties of rabbit testicular dipeptidyl carboxypeptidase.

Rabbit testicular dipeptidyl carboxypeptidase activity was purified by a procedure exploiting its affinity for N-alpha-[1-(S)-carboxy-3-phenylpropyl]-L-lysyl-L-proline. The molecular, catalytic, and immunological properties of the testicular enzyme are presented and compared with the corresponding properties of pulmonary angiotensin-converting enzyme. Although catalytically similar and immunologically related to pulmonary dipeptidyl carboxypeptidase, the testicular enzyme has a molecular weight (100,000) which is lower by a factor of about one-third and differs in its NH2 and COOH termini. Furthermore, we present evidence that the testicular enzyme is not a post-translation product of the pulmonary type enzyme. These data suggest that testicular and pulmonary dipeptidyl carboxypeptidase are two distinct proteins which are catalytically similar and immunologically closely related.