Development of cardiac form and function in ectothermic sauropsids

Evolutionary morphologists and physiologists have long recognized the phylogenetic significance of the ectothermic sauropsids. Sauropids have been classically considered to bridge between early tetrapods, ectotherms, and the evolution of endotherms. This transition has been associated with many modifications in cardiovascular form and function, which have changed dramatically during the course of vertebrate evolution. Most cardiovascular studies have focused upon adults, leaving the development of this critical system largely unexplored. In this essay, we attempt a synthesis of sauropsid cardiovascular development based on the limited literature and indicate fertile regions for future studies. Early morphological cardiovascular development, i.e., the basic formation of the tube heart and the major pulmonary and systemic vessels, is similar across tetrapods. Subsequent cardiac chamber development, however, varies considerably between developing chelonians, squamates, crocodilians, and birds, reflected in the diversity of adult ventricular structure across these taxa. The details of how these differences in morphology develop, including the molecular regulation of cardiac and vascular growth and differentiation, are still poorly understood. In terms of the functional maturation of the cardiovascular system, reflected in physiological mechanisms for regulating heart rate and cardiac output, recent work has illustrated that changes during ontogeny in parameters such as heart rate and arterial blood pressure are somewhat species‐dependent. However, there are commonalities, such as a β‐adrenergic receptor tone on the embryonic heart appearing prior to 60% of development. Differential gross morphological responses to environmental stressors (oxygen, hydration, temperature) have been investigated interspecifically, revealing that cardiac development is relatively plastic, especially, with respect to change in heart growth. Collectively, the data assembled here reflects the current limited morphological and physiological understanding of cardiovascular development in sauropsids and identifies key areas for future studies of this diverse vertebrate lineage. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Structural studies on a protein-binding zinc-finger domain of Eos reveal both similarities and differences to classical zinc fingers

The oligomerization domain that is present at the C terminus of Ikaros-family proteins and the protein Trps-1 is important for the proper regulation of developmental processes such as hematopoiesis. Remarkably, this domain is predicted to contain two classical zinc fingers (ZnFs), domains normally associated with the recognition of nucleic acids. The preference for protein binding by these predicted ZnFs is not well-understood. We have used a range of methods to gain insight into the structure of this domain. Circular dichroism, UV-vis, and NMR experiments carried out on the C-terminal domain of Eos (EosC) revealed that the two putative ZnFs (C1 and C2) are separable, i.e., capable of folding independently in the presence of Zn(II). We next determined the structure of EosC2 using NMR spectroscopy, revealing that, although the overall fold of EosC2 is similar to other classical ZnFs, a number of differences exist. For example, the conformation of the C terminus of EosC2 appears to be flexible and may result in a major rearrangement of the zinc ligands. Finally, alanine-scanning mutagenesis was used to identify the residues that are involved in the homo- and hetero-oligomerization of Eos, and these results are discussed in the context of the structure of EosC. These studies provide the first structural insights into how EosC mediates protein-protein interactions and contributes to our understanding of why it does not exhibit high-affinity DNA binding.     

Gait retraining to reduce the knee adduction moment through real-time visual feedback of dynamic knee alignment

Varus knee alignment is a risk factor for medial knee osteoarthritis and is associated with high knee adduction moments. Therefore, reducing the knee adduction moment in varus-aligned individuals with otherwise healthy knees may reduce their risk for developing osteoarthritis. A gait modification that improves dynamic knee alignment may reduce the adduction moment, and systematic training may lead to more natural-feeling and less effortful execution of this pattern. To test these hypotheses, eight healthy, varus-aligned individuals underwent a gait modification protocol. Real-time feedback of dynamic knee alignment was provided over eight training sessions, using a fading paradigm. Natural and modified gait were assessed post-training and after 1 month, and compared to pre-training natural gait. The knee adduction moment, as well as hip adduction, hip internal rotation and knee adduction angles were evaluated. At each training session, subjects rated how effortful and natural-feeling the modified pattern was to execute. Post-training, the modified pattern demonstrated an 8° increase in hip internal rotation and 3° increase in hip adduction. Knee adduction decreased 2°, and the knee adduction moment decreased 19%. Natural gait did not differ between the three visits, nor did the modified gait pattern between the post-training and 1 month visits. The modified pattern felt more natural and required less effort after training. Based on these results, gait retraining to improve dynamic knee alignment resulted in significant reductions in the knee adduction moment, primarily through hip internal rotation. Further, systematic training led to more natural-feeling and less effortful execution of the gait pattern.     

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

Peptide toxins found in a wide array of venoms block K⁺ channels, causing profound physiological and pathological effects. Here we describe the first functional K⁺ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23_TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23_TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K⁺ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K⁺ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K⁺ channels by co-localizing with and trapping MMP23_TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.     

Outcome of SurePath™ cervical samples reported as borderline nuclear change by cytological subtype and high‐risk HPV status

N. Gupta, N. Dudding, J. Crossley, S.J. Payyappilly and J.H.F. Smith
Outcome of SurePath? cervical samples reported as borderline nuclear changes by cytological subtype and high‐risk HPV status Background: The average borderline rate in cervical cytology samples for English laboratories was 3.8% with the range being 2.0–6.8% at the time of the present study, which was undertaken in order to determine the association between different subtypes of borderline nuclear change (BNC), high‐grade cervical intraepithelial neoplasia (CIN) and high‐risk human papillomavirus (hrHPV) status. Materials and methods: Of 68 551 SurePath^TM cervical samples reported in one laboratory over a period of 2 years, 2335 (3.4%) were reported as BNC. hrHPV status was known in 1112 cases (47.6%). The outcome was known only for women with hrHPV‐positive BNC, who were recommended for colposcopy under the National Health Service Cervical Screening Programme sentinel site protocol. Women with hrHPV‐negative BNC were returned to 3‐yearly recall. The cases were subdivided into BNC, high‐grade dyskaryosis cannot be excluded (B‐HG; 105 cases); BNC with koilocytosis (B‐K; 421 cases); BNC with other features of HPV (B‐HPV; 160 cases); and BNC, not otherwise specified (B‐NOS; 426 cases) and were correlated with the histological outcome where available. Results: The study population age ranged from 23 to 65 years. Cases that tested positive for hrHPV by Qiagen HCII assay comprised 78.1%, 81.0%, 73.1% and 67.8% of B‐HG, B‐K, B‐HPV and B‐NOS categories, respectively. CIN2 or worse (CIN2+) was found in 64.6%, 10.0%, 19.7% and 20.1% of hrHPV‐positive cases of B‐HG, B‐K, B‐HPV and B‐NOS, respectively, which was significantly higher in the B‐HG category (P < 0.001) and lower in the B‐K category compared with B‐NOS (p < 0.001) and B‐HPV (p = 0.006) respectively. CIN3+ comprised 55.6%, 6.3%, 26.3% and 19.1% of biopsies in the same categories, respectively. Conclusions: Subtyping BNC is useful, especially B‐K and B‐HG, which, respectively, had the lowest and highest rates of detection of both CIN2+ and CIN3+, confirming that koilocytosis is likely to be associated with transient HPV infection. Women with B‐HG should be referred to colposcopy in the absence of HPV triage.     

The D-diastereomer of ShK toxin selectively blocks voltage-gated K+ channels and inhibits T lymphocyte proliferation

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T(EM)); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K(d) 36 nm (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K(+) channels tested. D-allo-ShK inhibited human T(EM) cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-allo-ShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K(+) channel studies; all-d analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.     

Les génotypes responsables de mucoviscidose ou d’absence bilatérale des canaux déférents ABCD

Over the last decade, the genetic basis for CBAVD has been identified by its association with CFTR gene mutations, and CBAVD is now generally considered to be a mild or incomplete form of CF. In this review, the author summarizes the main results of compilation of CFTR gene analysis conducted in French laboratories for 3,923 patients with CF and 800 males with CABVD. The degree of clinical expression can be affected by several variables, including the molecular mechanisms by which the various CFTR mutations impair or disrupt the function of the CFTR chloride channel. Phenotypic expression of CFTR mutational genotypes varies from severe, progressive pulmonary disease with pancreatic insufficiency (CF-PI), to mild pulmonary disease with pancreatic sufficiency (PS) or singleorgan forms of “CFTR-opathies”. In CF, a total of 310 different CFTR mutations accounting for 94% of 7,846 CF alleles have generated almost 500 different genotypes, comprising 2 severe mutations in 88% of cases (CF-PI), one severe mutation in trans to a mild mutation in 11% (CF-PS), and 2 mild mutations in 1% of identified genotypes. In CBAVD, 137 mutations scattered over the whole gene were identified in 60% of 1,600 CBAVD alleles during the study. Among the 150 characterized mutational CFTR genotypes, compound heterozygosity was the rule, and the most frequent CBAVD combinations were ΔF508/5T (35%), ΔF508/other mutation (30%, including ΔF508/R117H-7T: 5,6%), and 5T/other mutation (17%). No combination of two severe mutations was found in CBAVD (0%); by contrast with the CF population, 88% of genotypes identified in CBAVD comprised a severe mutation in trans to a mild mutation, and 12% consisted of 2 mild mutations. A total of 22 genotypes were shared by both CF and CBAVD. The role of the 5T allele as a splicing variant with variable, incomplete disease penetrance in CBAVD is reviewed. Other haplotype backgrounds, such as the TG12 sequence and the M470V polymorphism, may influence CFTR splicing and/or function. This study confirms the high molecular heterogeneity of CFTR mutations in CBAVD and emphasizes the importance of extensive CFTR analysis in these patients. Longterm follow-up studies of CBAVD patients are necessary in order to predict the phenotypic consequences of numerous CFTR mutational genotypes.