External rotation of the femoral component increases asymmetry of the posterior condyles.

AIMS: The morphometry of the distal femur was largely studied to improve bone-implant fit in total knee arthroplasty (TKA), but little is known about the asymmetry of the posterior condyles. This study aimed to investigate the dimensions of the posterior condyles and the influence of externally rotating the femoral component on potential prosthetic overhang or under-coverage. PATIENTS AND METHODS: We analysed the shape of 110 arthritic knees at the time of primary TKA using pre-operative CT scans. The height and width of each condyle were measured at the posterior femoral cut in neutral position, and in 3º and 5º of external rotation, using both central and medial referencing systems. We compared the morphological characteristics with those of 14 TKA models. RESULTS: In the neutral position, the dimensions of the condyles were nearly equal. Externally rotating the femoral cut by 3º and 5º with 'central referencing' induced width asymmetry > 3 mm in 23 (21%) and 33 (30%) knees respectively, while with 'medial referencing' it induced width asymmetry > 3 mm in 43 (39%) and 75 (68%) knees respectively. The asymmetries induced by rotations were not associated with gender, aetiology or varus-valgus alignment. CONCLUSION: External rotation may amplify the asymmetry between the medial and lateral condyles, and exacerbate prosthetic overhang, particularly in the superolateral zone. 'Central referencing' guides result in less potential prosthetic overhang than 'medial referencing' guides. Cite this article: Bone Joint J 2017;99-B:894-903.

Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need?

Sequencing of the Arabidopsis genome revealed a unique complexity of the plant heat stress transcription factor (Hsf) family. By structural characteristics and phylogenetic comparison, the 21 representatives are assigned to 3 classes and 14 groups. Particularly striking is the finding of a new class of Hsfs (AtHsfC1) closely related to Hsf1 from rice and to Hsfs identified from frequently found expressed sequence tags of tomato, potato, barley, and soybean. Evidently, this new type of Hsf is well expressed in different plant tissues. Besides the DNA binding and oligomerization domains (HR-A/B region), we identified other functional modules of Arabidopsis Hsfs by sequence comparison with the well-characterized tomato Hsfs. These are putative motifs for nuclear import and export and transcriptional activation (AHA motifs). There is intriguing flexibility of size and sequence in certain parts of the otherwise strongly conserved N-terminal half of these Hsfs. We have speculated about possible exon-intron borders in this region in the ancient precursor gene of plant Hsfs, similar to the exon-intron structure of the present mammalian Hsf-encoding genes.

The balance of nuclear import and export determines the intracellular distribution and function of tomato heat stress transcription factor HsfA2.

Tomato heat stress transcription factor HsfA2 is a shuttling protein with dominant cytoplasmic localization as a result of a nuclear import combined with an efficient export. Besides the nuclear localization signal (NLS) adjacent to the oligomerization domain, a C-terminal leucine-rich motif functions as a nuclear export signal (NES). Mutant forms of HsfA2 with a defective or an absent NES are nuclear proteins. The same is true for the wild-type HsfA2 if coexpressed with HsfA1 or in the presence of export inhibitor leptomycin B (LMB). Fusion of the NES domain of HsfA2 to HsfB1, which is a nuclear protein, caused export of the HsfB1-A2NES hybrid protein, and this effect was reversed by the addition of LMB. Due to the lack of background problems, Chinese hamster ovary (CHO) cells represent an excellent system for expression and functional analysis of tomato Hsfs. The results faithfully reflect the situation found in plant cells (tobacco protoplasts). The intriguing role of NLS and NES accessibility for the intracellular distribution of HsfA2 is underlined by the results of heat stress treatments of CHO cells (41 degrees C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41 degrees C even in the presence of LMB. The temperature-dependent conformational transition of HsfA2 with shielding of the NLS evidently needs intramolecular interaction between the internal HR-A/B and the C-terminal HR-C regions. It is not observed with the HR oligomerization domain (HR-A/B region) deletion form of HsfA2 or in HsfA2-HsfA1 hetero-oligomers.

Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts.

Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat-stressed plant cells. We have developed an experimental system involving expression of cytosolic class I and class II small heat-stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG-like complexes. Class I and class II small Hsps formed class-specific dodecamers of 210-280 kDa, which, upon heat stress, were incorporated into HSG complexes. Interestingly, class II dodecamers alone could form HSG-like complexes (auto-aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment). By analysing C-terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced auto-aggregation and for recruitment of class I proteins. The class-specific formation of dimers as a prerequisite for oligomerization was analysed by the yeast two-hybrid system. In the presence of the endogenous (tobacco) set of heat-stress-induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Although other, more distantly related, members of the Hsp20 family, i.e. the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo-oligomers of 200-700 kDa, none of them could be recruited to HSG complexes.

Cytosolic heat-stress proteins Hsp17.7 class I and Hsp17.3 class II of tomato act as molecular chaperones in vivo.

Small heat-stress proteins (sHsps) are the most abundant stress-induced proteins with up to 20 different members in higher plants. In the cytoplasm, two different classes can be distinguished. Two cDNA clones from tomato Lycopersicon peruvianum (L.) Mill., each coding for one of the cytoplasmic sHsp subfamilies, were analyzed with respect to their transcript and protein expression, genome organization and chaperone activity. Neither type was present under control conditions but both appeared upon heat stress and in mature fruits. Expression of the class II transcript was found to be induced at slightly lower temperatures than the class I transcript. Protein analysis using class-specific antibodies revealed an identical expression pattern of both corresponding proteins. Transient expression in an Arabidopsis thaliana (L.) Heynh. cell culture showed that, despite the difference in their amino acid sequence, both classes are functionally active as chaperones in vivo, as shown by their ability to prevent thermal inactivation of firefly luciferase in a cellular environment.

The role of AHA motifs in the activator function of tomato heat stress transcription factors HsfA1 and HsfA2.

Using reporter assays in tobacco protoplasts and yeast, we investigated the function of the acidic C-terminal activation domains of tomato heat stress transcription factors HsfA1 and HsfA2. Both transcription factors contain short, essential peptide motifs with a characteristic pattern of aromatic and large hydrophobic amino acid residues embedded in an acidic context (AHA motifs). The prototype is the AHA1 motif of HsfA2, which has the sequence DDIWEELL. Our mutational analysis supports the important role of the aromatic and large hydrophobic amino acid residues in the core positions of the AHA motifs. The pattern suggests the formation of an amphipathic, negatively charged helix as the putative contact region with components of the basal transcription complex. In support of this concept, proline or positively charged residues in or adjacent to the AHA motifs markedly reduce or abolish their activity. Both AHA motifs of HsfA1 and HsfA2 contribute to activator potential, and they can substitute for each other; however, there is evidence for sequence and positional specificity.

The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules.

In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen. Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25 degrees C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules. The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1. When expressed in tobacco protoplasts by use of a transient-expression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1. The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners. Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test in Saccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

Intracellular distribution and identification of the nuclear localization signals of two plant heat-stress transcription factors.

Similar to heat-stress transcription factors (HSFs) from non-plant sources, HSFA1 and HSFA2 from tomato (Lycopersicon esculentum Mill) contain two conserved clusters of basic amino acid residues (K/R1 and K/R2) which might serve as nuclear localization signal (NLS) motifs. Mutation of either one of them and functional testing of the corresponding proteins in a transient expression assay using tobacco (Nicotiana plumbaginifolia L:) protoplasts gave the following results. Whereas K/R1, positioned in all HSFs at the C-terminus of the DNA-binding domain, had no influence on nuclear import, the K/R1 mutants were impaired in their interaction with the DNA (band-shift assays). In contrast to this, mutants of the K/R2 motif, found 15-20 amino acid residues C-terminal of the oligomerization domain (HR-A/B region), had wild-type activity in DNA-binding but were defective in nuclear import. Thus, for the related tomato HSFA1 and HSFA2 the K/R2 cluster represents the only NLS motif, and in this function it cannot be replaced by K/R1.

Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo.

Using Agrobacterium, we developed a method to transform an Arabidopsis cell suspension culture. A stably transformed cell line expressing high levels of firefly luciferase (Luc) was used for in vivo studies of thermal denaturation and renaturation of the enzyme and the protective role of different chaperones. Luc activity was monitored under heat stress and recovery conditions in control, thermotolerant cells and cells expressing plant chaperones after transient cotransformation with plasmids encoding proteins of the heat shock protein Hsp90, Hsp70, or Hsp20 family. The effects of the expressed proteins were specific. The Hsp17.6 class I protein maintained Luc activity on a level comparable with that observed in thermotolerant cells and improved Luc renaturation. Although transient expression of Hsp90 did not protect Luc from thermal denaturation, it accelerated Luc renaturation during recovery. In contrast to the other chaperones tested, overexpression of Hsp70 alone had no effect on denaturation and renaturation of Luc but enhanced Luc renaturation if coexpressed with Hsp17.6.

The Hsf world: classification and properties of plant heat stress transcription factors.

Based on the partial or complete sequences of 14 plant heat stress transcription factors (Hsfs) from tomato, soybean, Arabidopsis and maize we propose a general nomenclature with two basic classes, i.e. classes A and B each containing two or more types of Hsfs (HsfA1, HsfA2 etc.). Despite some plant-specific peculiarities, essential functional domains and modules of these proteins are conserved among plants, yeast, Drosophila and vertebrates. A revised terminology of these parts follows recommendations agreed upon among the authors and representatives from other laboratories working in this field (see legend to Fig. 1). Similar to the situation with the small heat shock proteins (sHsps), the complexity of the hsf gene family in plants appears to be higher than in other eukaryotic organisms.

Solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24.

Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24. Samples of uniformly 15N-labeled and 15N, 13C-labeled recombinant proteins were used in the investigation. A near-complete assignment of the backbone 1H, 15N, and 13C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional 15N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments, 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-15N-edited and 13C-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations. The resulting three-dimensional structure contains a three-helix bundle and a small four-stranded antiparallel beta-sheet that forms a hydrophobic core. The two C-terminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding. In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between beta3 and beta4. This leads to a tight turn between these beta-strands.

Promoter specificity and deletion analysis of three heat stress transcription factors of tomato.

Transient expression assays in transformed tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts were used to test the activity of three tomato heat stress transcription factors, HSF24, HSF8 and HSF30, in a trans-activation and a trans-repression assay. The results document differences between the three HSFs with respect to their response to the configuration of heat stress promoter elements (HSEs) in the reporter construct (promoter specificity) and to the stress regime used for activation. Analysis of C-terminal deletions identified acidic sequence elements with a central tryptophan residue, which are important for HSF activity control. Surprisingly, heterologous HSFs from Drosophila and human cells, but not from yeast, were also functional as heat stress-induced transcription factors in this tobacco protoplast system.

HSP68--a DnaK-like heat-stress protein of plant mitochondria.

A 68-kDa heat-stress protein (HSP68) has been purified from cell-suspension cultures of tomato (Lycopersicon peruvianum L.). Antibodies raised against HSP68 cross-react with the Escherichia coli heat-stress protein DnaK. HSP68 was found to be a hydrophilic, ATP-binding protein. Immunological analysis of subcellular fractions and immunogold-labelling of ultrathin sections showed consistently that HSP68 is localized in the mitochondrial matrix. In-vitro translation experiments indicated that HSP68 is synthesized as a precursor protein. Immunoscreening of cDNA libraries from tomato and potato (Solanum tuberosum L.) led to the isolation of corresponding cDNA clones. The deduced amino-acid sequences show strong relationships to the DnaK-like proteins from bacteria and organelles of eukaryotic cells. The protein HSP68 is constitutively expressed, but its synthesis is increased during heat stress in all cells of higher plants investigated so far.