Protein engineering by expressed protein ligation.

By allowing the controlled assembly of synthetic peptides and recombinant polypeptides, expressed protein ligation permits unnatural amino acids, biochemical probes, and biophysical probes to be specifically incorporated into semisynthetic proteins. A powerful feature of the method is its modularity; once the reactive recombinant pieces are in hand and the optimal ligation conditions have been developed, it is possible to quickly generate an array of semisynthetic analogs by simply attaching different synthetic peptide cassettes--in most cases the synthetic peptides will be small and easy to make. From a practical perspective, the rate-determining step in the process is usually not the ligation step (it is based on a simple and efficient chemical reaction), but rather the generation of the reactive polypeptide building blocks. In particular, optimizing the yields of recombinant polypeptide building blocks can require some initial effort. However, it should be noted that the initial investment in time required to optimize the production of the recombinant fragment is offset by the ease and speed with which one can produce the material thereafter. In the example described in this chapter, the yield of soluble intein fusion protein was slightly better using the GyrA intein than for the VMA intein, although in both cases significant amounts of fusion protein were present in the cell pellet. Studies are currently underway to identify optimal refolding conditions for GyrA fusion proteins solubilized from inclusion bodies.

Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils.

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.

Introduction of unnatural amino acids into proteins using expressed protein ligation.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.

Distinct isoforms of chicken decorin contain either one or two dermatan sulfate chains.

Decorin, a member of a family of proteins with leucine-rich repeat motifs, is a widely distributed extracellular matrix proteoglycan that is thought to be responsible for the structure, tissue organization, and surface properties of fibrils. In mammals, decorin carries one chondroitin/dermatan sulfate chain as a distinction from its homologue, biglycan, which contains two glycosaminoglycan chains. With the aim to study decorin-collagen interactions in chicken, where the fibrillar organization of cartilage collagens is best understood, we have isolated decorin-related proteoglycans from sternal cartilage of 40-day-old broiler chickens. Small chondroitin/dermatan sulfate proteoglycans were resolved by hydrophobic interaction chromatography into two fractions, DCN I and DCN II. Both forms contained dermatan sulfate and, in addition, keratan sulfate chains. Tryptic fingerprinting revealed that the core proteins of DCN I and DCN II were identical. The protein was identified as decorin by amino-terminal sequencing. DCN II was found to contain two dermatan sulfate chains, whereas DCN I had a single dermatan sulfate chain. The dermatan sulfate attachment sites are located near the NH2 terminus of the core protein, i.e. at Ser-4 and Ser-16 in DCN II and at Ser-4 in DCN I. The keratan sulfate attachment sites are located in the central portion of the core protein, at Asn-179 and Asn-230. The presence of two dermatan sulfate chains renders the chicken proteoglycan DCN II structurally similar to mammalian biglycan. Interestingly, biglycan has not been detected in chicken. Therefore, in birds, DCN II may function as a biglycan substitute.

Foreign body in upper airway. Unsuspected cause of obstruction.

Airway obstruction from a foreign body is usually an acute life-threatening event. In this case, a complete upper dental plate was the unsuspected cause of intermittent respiratory distress. The authors discuss the clinical and laboratory findings and suggest ways to prevent unnecessary delays in diagnosis and treatment.

[Status of computerized tomography for the diagnosis of diseases of the gallbladder and bile ducts]. / Stellenwert der Computertomographie für die Diagnostik von Erkrankungen der Gallenblase und Gallenwege.

After a short introduction to the computed tomographic examination technique and the demonstrability of the anatomic structures in the section picture the findings in cholelithiasis, cholecystitis and cholangitis, in the malignoma of the biliary system as well as of the traumatic lesion of the bile ducts are described. In the diseases mentioned the reliability of the computed tomography is high and it does not fall behind the sonography. Nevertheless, the sonography is primarily to be used on account of missing radiation exposure, smaller costs and broader spread of appliances. Then the computed tomography is performed in selected cases which remain unclear such as in complicated lithiases and in the suspicion of malignoma.

[Carcinoid tumor with cystic liver metastases]. / Karzinoid mit zystischen Lebermetastasen.

Generally metastases of carcinoid tumours are pseudocystic in nearly 10% of the patients. In our case the guiding diagnostic importance of sonography and computed tomography could be proved. Results have to be completed by histologic findings and hormonal analysis (serotonin, 5-hydroxyindol acetic acid). - Morphological signs especially of the new imaging techniques are often a supposition for the modern therapy, e.g. the application of serotonin antagonists, local perfusion of cytostatic substances and if possible transplantation of the liver.

[Ablative hormone therapy and chemotherapy of metastatic breast cancer]. / Ablative Hormontherapie und Chemotherapie beim metastasierenden Mammakarzinom.

Case report of a 45-year-old woman with breast cancer who underwent mastectomy with axillary lymphonodectomy and radiation therapy. Two years after primary therapy liver metastases have been proved by sonography, liver scintigraphy and computerized tomography scanning. A nearly complete remission was achieved by ablative hormone therapy and CMF-chemotherapy.

Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes.

A novel ADP-ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and heat treatment. The molecular mass of botulinum ADP-ribosyltransferase C3 was found to be 25 kDa. In the presence of [32P]NAD but not with [carbonyl-14C]NAD, C3 labelled 21-24-kDa protein(s) in membranes of human platelets and other tissues. The Km value of the ADP-ribosylation reaction for NAD was about 2 microM. Labelling of the 21-24-kDa protein(s) by C3 was largely reduced by addition of nicotinamide. Snake venom phosphodiesterase cleaved the ADP-ribose attached to the 21-24-kDa protein(s) by C3 and released 5'AMP. C3 catalyzed hydrolysis of [carbonyl-14C]NAD and released [carbonyl-14C]nicotinamide. ADP-ribosylation of 21-24-kDa platelet membrane protein(s) was biphasically regulated by Mg2+, Mn2+ and Ca2+. In the absence of free divalent cations GTP, GTP[gamma S] and GDP but not GDP[beta S], GMP, ATP or ATP[gamma S] increased labelling by C3. In the presence of Mg2+, GTP[gamma S] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[gamma S] = GTP = GDP greater than GDP[beta S] greater than GMP much greater than ATP = GMP = ATP[gamma S]. The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21-24-kDa GTP-binding protein(s).

[Complaints following cholecystectomy]. / Beschwerden nach Cholezystektomie.

A subdivision of the most frequent complaints after cholecystectomy is given. For the clarification of the manifold causes of these inhomogeneous pictures of complaints a considerable diagnostic expenditure is necessary, in which case the importance of ERCP is particularly emphasized. In concrements of the choledochus as well as in short-distance papillary stenoses nowadays above all the endoscopic papillotomy is therapeutically taken into consideration. Recommendations are given for the medicamentous treatment of functional complaints.

[Possibilities and limitations of gastrointestinal radiography]. / Möglichkeiten und Grenzen gastrointestinaler Röntgendiagnostik.

The picture intensifier television radioscopy, picture intensifier initial level photography, double contrast method and pharmacoradiography together with endoscopy further the optimation of the diagnostics of the diseases of oesophagus, stomach and intestine. The value of the X-ray diagnostics for the recognition of the biliary diseases and of the incleared jaundice is high. In diseases of liver and pancreas the X-ray diagnostics may further the finding of decision. Ways for a better interdisciplinary cooperation are demonstrated.

[Relation of radiographic and clinical findings to the pathomorphological changes in Wegener's granulomatosis]. / Beziehungen röntgenologischer und klinischer Befunde zu pathomorphologischen Veränderungen bei Wegenerscher Granulomatose.

On the basis of two own observations the clinical and radiological routine methods undergo a critical judgment concerning their valency for the diagnosis of Wegener's granulomatosis. The two patients showed the picture of a severe septical disease with high fever and expressed inflammatory changes of the biological reactions. In the first observation a pulmonary round focus and terminally the renal failure, in the second case severe inflammatory changes of the lungs without response to antibiotic therapy were in the foreground. The appearance of this disease as generalized vasculitis is conditioned by the manifold clinical symptomatology and thus renders the diagnostics extraordinarily difficult. The simultaneous affection of the nasopharyngeal space, of the lung and the kidneys may be a guide for the diagnosis. The X-ray findings of the lung are not able to found sufficiently the diagnosis.

[Intramural diverticulosis and adenomyomatosis of the gallbladder]. / Zur intramuralen Divertikulose und Adenomyomatose der Gallenblase.

Issuing from an own observation the clinical picture of the adenomyomatosis and the intramural diverticulosis of the gall bladder is considered from the clinical, radiological and pathologo-anatomical point of view. The opinions which are not homogeneous are explained and discussed. Also abortive forms may radiologically be proved with probability.

[Pulmonary metastasizing of prostatic carcinoma--source of errors in the ambulatory x-ray examination of lungs]. / Die pulmonale Metastasierung des Prostatakarzinoms--eine Fehlerquelle bei der ambulanten röntgenologischen Lungenuntersuchung.

It is reported on eight patients with a primary carcinoma of the prostatic gland who were admitted to the hospital for clinical clarification of conspicuous pulmonary findings. The radiological findings of a general radiograph of the thorax are multiform and for themselves alone they do not permit an etiological coordination. For this reason the demand can be derived in patients of the adequate age group with tumour symptomatology can also be thought of a carcinoma of the prostatic gland and already during the examination in the outpatient department to carry out a palpatory examination and, if possible, to make a general radiograph of spinal column and pelvis, since in these cases the diagnosis can be made already before admission.

[Deep duodenoscopy and endoscopic retrograde cholangiopancreaticography]. / Tiefe Duodenoskopie und endoskopische retrograde Cholangiopankreatikographie

A survey is given on indications, results and complications of the deep duodenoscopy and endoscopic retrograde cholangiopancreaticography. The endoscopic retrograde pancreatography is indicated in the chronic forms of pancreatitis, in suspicion to cysts of the pancreas or tumours. Its results have a decisive influence on the indication to surgical procedure and on the planning of the intervention. The endoscopic retrograde cholangiography is of greatest practical significance for the differential diagnosis of the cholestatic icterus: non-obstructed bile ducts exclude an extrahepatic icterus and render a laparotomy useless. Furthermore, the endoscopic retrograde cholangiography is indicated when the demonstration of the duct is intravenously insufficient, when papillary or prepapillary narrowings are present and in the etiologically unclear syndrome of postcholecystectomy. The most dangerous complications of the endoscopic retrograde pancreatography are the acute pancreatitis and the infection of cysts. Cholangitides and septicaemias appear after the endoscopic retrograde cholangiography only in such cases when obstructions of the drainage are present.