[Primary revision with replasty of the anterior cruciate ligament]. / Die primäre Revision mit Replastik des vorderen Kreuzbandes.

OBJECTIVE: Restoration of knee stability after rerupture of an anterior cruciate ligament (ACL) graft. INDICATION: Acute and chronic functional instability with rerupture of an ACL graft with subjective instability with anatomical or non-anatomical bone tunnel without tunnel widening. CONTRAINDICATIONS: Partial anatomical bone tunnels of the previous operation, significant tunnel widening of anatomical bone tunnels, local infection of the knee joint, local soft tissue damage. SURGICAL TECHNIQUE: Graft choices are hamstring tendons (semitendinosus muscle, gracilis muscle), the quadriceps tendon, patellar tendon and a peroneus tendon split graft. In cases with anatomical tunnels, careful debridement is performed down to the tunnel wall. In non-anatomical tunnels, a new femoral tunnel is drilled over a deep anteromedial portal with the knee flexed more than 110° in the insertion area of the ACL. Using drills and dilators, a tunnel is prepared. At the tibia, the anterior horn of the lateral meniscus serves as a landmark in the absence of an ACL stump. The cortical tibial tunnel aperture is probed with a guide wire and the tunnel is drilled stepwise until the tunnel wall is reached, which is debrided with a spoon or synovial resector to remove graft residues and implants from the tunnel. The femoral fixation can either be done with a flip button, an interference screw or in the case of a bone block graft implant-free. At the tibial side, the graft is fixed with a resorbable interference screw and fixation button. POSTOPERATIVE MANAGEMENT: The rehabilitation program comprises 4-5 phases. Inflammatory phase (weeks 1-2): control of pain and swelling (cooling, isometric tension exercises, 20 kg partial load). Phase 2 (weeks 2-6): increasing load and range of motion with closed chain exercises (target: extension/flexion 0-0-120°). Phase 3 (from week 6): strength and coordination exercises. Phase 4: balance, strength and jump exercises. Return to competitive sport not before postoperative month 6-10. RESULTS: Included were 51 patients with recurrent instability after ACL surgery where primary ACL replacement was performed with ipsilateral bone quadriceps tendon graft or contralateral semitendinosus-gracilis graft. All patients had anatomical or non-anatomical tunnel locations without significant widening (>11 mm). After 2 years, the side-to-side difference for anterior tibial translation measured with the KT 1000 arthrometer was 2.0 ± 1.2 mm for the quadriceps group and 3.0 ± 2.9 mm for the semitendinosus-gracilis group (P = 0.461). No difference in the rate of positive pivot shift tests (P = 0.661); no significant difference in the individual Knee Injury and Osteoarthritis Outcome Score (KOOS) subscores or in the frequency of anterior knee pain.

STEM Analysis of Caenorhabditis elegans muscle thick filaments: evidence for microdifferentiated substructures.

In the thick filaments of body muscle in Caenorhabditis elegans, myosin A and myosin B isoforms and a subpopulation of paramyosin, a homologue of myosin heavy chain rods, are organized about a tubular core. As determined by scanning transmission electron microscopy, the thick filaments show a continuous decrease in mass-per-length (MPL) from their central zones to their polar regions. This is consistent with previously reported morphological studies and suggests that both their content and structural organization are microdifferentiated as a function of position. The cores are composed of a second distinct subpopulation of paramyosin in association with the alpha, beta, and gamma-filagenins. MPL measurements suggest that cores are formed from seven subfilaments containing four strands of paramyosin molecules, rather than the two originally proposed. The periodic locations of the filagenins within different regions and the presence of a central zone where myosin A is located, implies that the cores are also microdifferentiated with respect to molecular content and structure. This differentiation may result from a novel "induced strain" assembly mechanism based upon the interaction of the filagenins, paramyosin and myosin A. The cores may then serve as "differentiated templates" for the assembly of myosin B and paramyosin in the tapering, microdifferentiated polar regions of the thick filaments.

Formulation of sustained release aqueous Zn-hirudin suspensions.

Sustained release formulations for recombinant hirudin (rHir), an anticoagulant thrombin-specific inhibitor, were developed. Zn-rHir suspensions were formed by precipitation with zinc salts at neutral pH. Characterization of protein precipitation was by UV analysis, capillary electrophoresis (CE), zinc analysis, light and electron microscopy, and particle size analysis. The precipitation of aqueous rHir solution with ZnCl(2) solution at neutral pH resulted in Zn-rHir suspensions. Optimum yields of pelletized Zn-rHir were obtained between pH 7.0 and 7.4. For complete precipitation ( approximately 100%) a molar ratio of zinc to rHir of >28 was necessary. As shown by electron microscopy, the smallest resolvable unit of Zn-rHir suspensions was 20 nm. Agglomerates of up to 200 microm were observed by light microscopy. Zinc salt-induced precipitation phenomena were also investigated using ZnBr(2), ZnI(2), Zn(NO(3))(2) and ZnSO(4) instead of ZnCl(2). ZnSO(4) showed the lowest precipitation efficiency. All other salts behaved similar to ZnCl(2). Upon storage the pelletized protein content of the ZnCl(2) based precipitates was stable ( approximately 95% rHir after 1 year at room temperature), whereas the pelletized protein content of ZnSO(4) based precipitates dropped sharply after precipitation (2% remaining after 13 days at room temperature). This indicates a transition of the ZnSO(4) based precipitates to hexagonal basic zinc sulfate plates and free rHir. The driving force is the lower aqueous solubility of basic zinc sulfate as compared to the higher solubility of basic zinc chloride.

The intermediate filament protein consensus motif of helix 2B: its atomic structure and contribution to assembly.

Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central alpha-helical rod domain. We have analyzed its contribution to the various stages of assembly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them with the wild-type and tailless proteins. It is surprising that in buffers of low ionic strength and high pH where the full-length proteins form tetramers, both VimIAT and DesIAT associated into various high molecular weight complexes. After initiation of assembly, both VimIAT and DesIAT aggregated into unit-length-type filaments, which rapidly longitudinally annealed to yield filaments of around 20 nm in diameter. Mass measurements by scanning transmission electron microscopy revealed that both VimIAT and DesIAT filaments contained considerably more subunits per cross-section than standard intermediate filaments. This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control of filament width, rather than elongation, during assembly. To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amino-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation. The atomic structure shows that starting from Tyr400 the two helices gradually separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis.

Structural analysis of F18 fimbriae expressed by porcine toxigenic Escherichia coli.

The F18 fimbriae expressed by porcine toxigenic Escherichia coli strains are 1- to 2-mm-long filaments that mediate the adhesion of the bacteria to enterocytes. The backbone of these fimbriae is built from a major structural 15.1-kDa protein, FedA. The structure of isolated negatively stained F18 fimbriae imaged by dark-field scanning transmission electron microscopy (STEM) was resolved to approximately 2 nm. Analyzing their helical symmetry showed the axially repeating units to alternate in a "zigzag" manner around the helical axis with an axial rise of 2.2 nm. Two repeating units give rise to the observed 4.3-nm helical repeat, which is practically identical to the pitch of the one-start helix formed. Additionally, an axially repeating pattern with a 27-nm spacing was found on rotary-shadowed fimbriae. Mass-per-length determination of unstained F18 fimbriae by STEM revealed the axially repeating unit to have a molecular mass of 25.4 kDa, indicating that it is a FedA monomer, with the difference in mass arising from the minor subunits, FedE and FedF. The presence of the latter two proteins might cause the observed 27-nm axial pattern.

Purification and analysis of authentic CLIP-170 and recombinant fragments.

We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170. Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is alpha-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced approximately 37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubules in vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170. These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.

Interactions between coiled-coil proteins: Drosophila lamin Dm0 binds to the bicaudal-D protein.

In a yeast two-hybrid screen we identified an interaction between Drosophila lamin Dm0, a structural nuclear protein, and BICD, a protein involved in oocyte development. The interaction can be reconstituted in vitro and takes place between segments of both proteins predicted to form coiled coils. The affinity for lamin Dm0 of the minimal binding site on BICD is modulated in a complex fashion by other BICD segments. A point mutation, F684I, that causes the dominant, bicaudal, Bic-D phenotype inhibits lamin binding in the context of the minimal lamin-binding site, but not in a larger BICD fragment. The minimal lamin-binding site of BICD binds to a few other coiled-coil proteins, but binding to these proteins is not influenced by the F684I point mutation, suggesting that the interaction with lamin may play a role in Bic-D function. Our structural studies demonstrated that BICD is 60-70% alpha-helical, is a dimer, and consists of two parts: a thin rod-shaped part of about 32 nm, and a thicker rod-shaped part of about 26 nm. Likely, the thinner rod-shaped part of full-length BICD consists of the N-terminal half of the protein, and the lamin-binding site is located within the thicker rod-shaped part.

Characterization of distinct early assembly units of different intermediate filament proteins.

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

Fine structural and functional consequences of deglycosylation of the platelet adhesion receptor GPIb-IX (CD 42b).

To investigate the role of the glycosylation of the platelet receptor glycoprotein Ib (GPIb, CD 42b), platelets and purified GPIb were deglycosylated by neuraminidase, O- and N-glycosidases. N-deglycosylation and neuraminic-acid cleavage had little effect on ristocetin and botrocetin-induced platelet agglutination. However, O-deglycosylation reduced the response by approximately 50%, and total deglycosylation (the combination of all three glycosidases) fully abolished the response to ristocetin. Interestingly, binding of von Willebrand Factor (vWF) to purified GPIb in the presence of ristocetin and botrocetin in a standardized microtiter plate assay was not altered by partial or even by total deglycosylation. Electron microscopy indicated that the normally stretched approximately 50 nm long molecule was approximately 32 nm after N-deglycosylation, approximately 20 nm after O-deglycosylation, and reduced in a approximately 15 nm long collapse by total deglycosylation. These results suggest that deglycosylation has major structural impacts on GPIb, strongly impairing platelet-vWF interactions; however, vWF binding to isolated GPIb remains unaffected.

Structure of a filamentous phosphoglycoprotein polymer: the secreted acid phosphatase of Leishmania mexicana.

The insect stage of the protozoan parasite Leishmania mexicana secretes a filamentous acid phosphatase (secreted acid phosphatase, SAP), a polymeric phosphoglycoprotein. The wild-type (wt) SAP filament is a copolymer composed of two related gene products SAP1 and SAP2, which are identical in the enzymatically active NH2-terminal domain and the COOH-terminal domain, but differ in the length of a highly glycosylated Ser/Thr-rich repeat region (32 amino acids and 383 amino acids, respectively) which is located between these domains. When expressed separately, full length SAP1, SAP2, or the NH2-terminal domain alone, are able to assemble into filaments. The Ser/Thr-rich region is the exclusive target for a novel type of O-glycosylation via phosphoserines. By using glycerol spraying/low-angle rotary metal shadowing and labelling with monoclonal antibodies it is demonstrated that the repetitive region adopts an extended conformation forming side arms which project radially from the filament core and terminate with the COOH-terminal domain. The length of the side arms of SAP1 and SAP2 (20 nm and 90 nm, respectively) corresponds to the predicted length of the Ser/Thr-rich repeat region of SAP1 and SAP2. Mass determination by scanning electron microscopy (STEM) shows that one morphologically defined globular particle of the filament core is a polypeptide dimer. We propose a model for the filament core, in which the globular NH2-terminal SAP domains form one strand composed of polypeptide dimers or two tightly associated strands of monomers which may twist into a double helix, similar to actin filaments. The highly O-glycosylated side arms project from the filament core conferring an overall bottle-brush-like appearance. The L. mexicana SAP is compared to SAPs secreted by the closely related species L. amazonensis and L. donovani.

Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments.

The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded alpha-helical coiled coil formation of their approximately 350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct approximately 56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a approximately 72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a approximately 310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, approximately 24.5 nm intervals, thereby giving them a "beaded" appearance with an axial periodicity of about 24.5 nm, which is approximately 3 nm longer than the corresponding approximately 21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.

Low frequency of BRCA1 germline mutations in 45 German breast/ovarian cancer families.

In this study we investigated 45 German breast/ovarian cancer families for germline mutations in the BRCA1 gene. We identified four germline mutations in three breast cancer families and in one breast-ovarian cancer family. among these were one frameshift mutation, one nonsense mutation, one novel splice site mutation, and one missense mutation. The missense mutation was also found in 2.8% of the general population, suggesting that it is not disease associated. The average age of disease onset in those families harbouring causative mutations was between 32.3 and 37.4 years, whereas the family harbouring the missense mutation had an average age of onset of 51.2 years. These findings show that BRCA1 is implicated in a small fraction of breast/ovarian cancer families suggesting the involvement of another susceptibility gene(s).

The human alpha-type proteasomal subunit HsC8 forms a double ringlike structure, but does not assemble into proteasome-like particles with the beta-type subunits HsDelta or HsBPROS26.

The eukaryotic proteasome is a barrel-shaped protease complex made up of four seven-membered rings of which the outer and inner rings may contain up to seven different alpha- and beta-type subunits, respectively. The assembly of the eukaryotic proteasome is not well understood. We cloned the cDNA for HsC8, which is one of the seven known human alpha-type subunits, and produced the protein in Escherichia coli. Recombinant HsC8 protein forms a complex of about 540 kDa consisting of double ringlike structures, each ring containing seven subunits. Such a structure has not earlier been reported for any eukaryotic proteasome subunit, but is similar to the complex formed by the recombinant alpha-subunit of the archaebacterium Thermoplasma acidophilum (Zwickl, P., Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765-770). The ability of HsC8 to form alpha-rings suggests that these complexes may play an important role in the initiation of proteasome assembly in eukaryotes. To test this, we used two human beta-type subunits, HsBPROS26 and HsDelta. Both these beta-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the alpha- and beta-subunit of T. acidophilum, coexpression of the human beta-type subunits with HsC8 does not result in the formation of proteasome-like particles, which would be in agreement with the notion that proteasome assembly in eukaryotes is much more complex than in archaebacteria.

In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

BRCA1 and BRCA2 mutation analysis in 86 early onset breast/ovarian cancer patients.

Eighty-six women fulfilling specific selection criteria were studied for germline mutations in two breast cancer susceptibility genes, BRCA1 and BRCA2, using the protein truncation test (PTT). Nine germline mutations were identified, six in BRCA1 and three in BRCA2. Of the six BRCA1 mutations, three have previously been described and three are new, and for BRCA2, one is a new mutation and the other two appear to occur at a site that has been described several times. Four kindreds were breast cancer families, one a breast/ovarian cancer family, and the sixth an ovarian cancer family. The three kindreds with BRCA2 mutations were classified as one breast/ovarian cancer family, one breast cancer family, and one family which harboured one early onset breast cancer patient and two melanoma patients. The mutations in BRCA1 were either insertions, deletions, or transitions which all resulted in a premature stop codon. Mutations in BRCA2 were all frameshift mutations as a result of either 2 or 4 bp deletions. Two BRCA2 mutations were identical, suggesting a Swiss founder effect which was confirmed by haplotype sharing. The 10% mutation detection rate is compatible with the relaxed criteria used for patient selection. Considering the relative ease with which coding sequences can be screened by PTT, this assay is useful as a first screen for BRCA1 and BRCA2 mutations.

Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains.

We have investigated the functional role of the non-helical end domains of vimentin on its assembly properties using truncated Xenopus and human recombinant proteins. Removal of the amino-terminal "head" domain yielded a molecule that did not assemble into 10 nm filaments but remained in a soluble oligomeric particle form with a sedimentation coefficient considerably smaller than that of wild-type vimentin (Vim(wt)). In contrast, removal of the carboxy-terminal "tail" domain had no obvious effect on the sedimentation characteristics. In particular, sedimentation equilibrium analysis under low ionic strength conditions yielded oligomeric particle species of Mr 135,000 to 360,000, indistinguishable from those obtained with Vim(wt). When induced to form filaments from this state by rapid dilution into filament forming buffer, Vim(wt) and Vim(deltaT) protein generated similar viscosity profiles. However, as determined by scanning transmission electron microscopy, under these conditions Vim(deltaT) formed filaments of heterogeneous diameter, corresponding to various distinct mass-per-length (MPL) values: whereas Vim(wt) yielded MPL values peaking between 40 and 45 kDa/nm, Vim(deltaT) filaments produced histograms which could be fitted by three Gaussian curves peaking between 37 and 131 kDa/nm. In contrast, when dialyzed against, instead of being rapidly diluted into, filament forming buffer, Vim(deltaT) gave histograms with one major peak at about 54 kDa/nm. The MPL heterogeneity observed for Vim(deltaT) was already evident at the earliest stages of assembly. For example, ten seconds after initiation, "unit-length" filament segments (58 to 63 nm) were formed with both wt and deltaT proteins, but the diameters were considerably larger for Vim(deltaT) compared to Vim(wt) (20(+/- 3) nm versus 16(+/- 3)nm), indicating a distinct role of the carboxy-terminal tail domain in the width control during unit-length filament formation. Despite this difference both Vim(deltaT) and Vim(wt) filaments appeared to grow stepwise in a modular fashion from such unit-length filament segments. This suggests that assembly occurred by a principally similar mechanism involving the end-on-fusion or annealing of unit-length filaments.

BRCA1 mutations in a selected series of breast/ovarian cancer patients.

Germline mutations in the BRCA1 gene have been associated with familial breast/ ovarian cancer in large families showing high penetrance of the disease. Little is known, however, about the contribution of BRCA1 mutations to breast/ovarian cancer in small families with few affected members or in isolated early onset cases. Therefore we examined the BRCA1 gene in 63 breast/ovarian cancer patients who either came from small families with as few as one affected first degree relative, or in patients who had no family history but had developed breast cancer under 40 years of age. Using the protein truncation test, we were able to identify three unique BRCA1 germline mutations (4.8%). Two of the probands had only one affected first degree and several second degree relatives and the third had three affected first degree relatives including two sisters who, when tested, were also found to carry the mutation. There was no family history of ovarian cancer in any of the three families.

Correlation between the development of extracolonic manifestations in FAP patients and mutations beyond codon 1403 in the APC gene.

The APC gene was investigated in 31 unrelated polyposis coli families by SSCP analysis and the protein truncation test. Twenty-three germline mutations were identified which gave rise to a variety of different phenotypes. Some of these mutations have already been described; however we report six previously unpublished mutations. Typical disease symptoms were observed in families who harboured mutations between exon 4 (codon 169) and codon 1393 of exon 15. Mutations beyond codon 1403 were associated with more varied phenotype with respect to the development of extracolonic symptoms. In this report we provide support for the notion that there appears to be a correlation between the location of an APC mutation (beyond codon 1403) and extracolonic manifestations of familial adenomatous polyposis.

Mechanism of the long tail-fiber deployment of bacteriophages T-even and its role in adsorption, infection and sedimentation.

Models for the tail-fiber deployment of T-even bacteriophages have been experimentally tested by correlating sedimentation constants, adsorption rates, protease inactivation kinetics, and fiber configurations of individual phages observed by electron microscopy. Neither the collective nor the individualistic model, i.e. coordinated fiber retraction and expansion or oscillation of fibers independently of each other, respectively, could satisfactorily account for the results presented. We propose a new intermediary model, in which the base-plate determines a collective behaviour by fixing the hinge angle, around which individual fibers oscillate freely. The bidisperse, so-called dual sedimentation was shown to occur mainly with nascent high-concentration phage stocks in potassium glutamate containing media. Indeed, when mature intracellular phages are released in 0.5 M potassium glutamate--a condition simulating the intracellular environment--only the fast form appears. Upon storage in the cold or release into 0.5 M chloride, both forms appear. Results confirming that the sedimentation constants of the fast and slow form roughly correspond to those of the monodisperse sedimentation, characteristic of the extreme pH values, i.e. 5 and 8, do not allow to conclude that fiber configuration is the only cause of the bidisperse sedimentation.