Combined myofibrillar and mitochondrial creatine kinase deficiency impairs mouse diaphragm isotonic function

Creatine kinase (CK) is an enzyme central to cellular high-energy phosphate metabolism in muscle. To characterize the physiological role of CK in respiratory muscle during dynamic contractions, we compared the force-velocity relationships, power, and work output characteristics of the diaphragm (Dia) from mice with combined myofibrillar and sarcomeric mitochondrial CK deficiency (CK[-/-]) with CK-sufficient controls (Ctl). Maximum velocity of shortening was significantly lower in CK[-/-] Dia (14.1 +/- 0.9 Lo/s, where Lo is optimal fiber length) compared with Ctl Dia (17.5 +/- 1.1 Lo/s) (P < 0.01). Maximum power was obtained at 0.4-0.5 tetanic force in both groups; absolute maximum power (2,293 +/- 138 W/m2) and work (201 +/- 9 J/m2) were lower in CK[-/-] Dia compared with Ctl Dia (2,744 +/- 146 W/m2 and 284 +/- 26 J/m2, respectively) (P < 0.05). The ability of CK[-/-] Dia to sustain shortening during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load) was markedly impaired, with CK[-/-] Dia power and work declining to zero by 37 +/- 4 s, compared with 61 +/- 5 s in Ctl Dia. We conclude that combined myofibrillar and sarcomeric mitochondrial CK deficiency profoundly impairs Dia power and work output, underscoring the functional importance of CK during dynamic contractions in skeletal muscle.     

Synthesis and antitumor evaluation of 2,5-disubstituted-indazolo[4, 3-gh]isoquinolin-6(2H)-ones (9-aza-anthrapyrazoles)

The synthesis and antitumor evaluation of 2, 5-disubstituted-indazolo[4,3-gh]isoquinolin-6(2H)-ones (9-aza-APs) are described. The key intermediates in the synthesis are benz[g]isoquinoline-5,10-diones which are substituted at positions 6 and 9 with groups of different nucleofugacity for SNAr displacements. The initial displacement of fluoride by a substituted hydrazine leads to the pyrazole analogues. Substitution of the remaining leaving group by an amine or BOC-protected amines leads to the 9-aza-APs 12. These analogues were converted into their maleate or hydrochloride salts 13. In two cases, namely, 13x and 13z, sidearm buildup was also employed in the synthetic pathway. In vitro evaluation of 9-aza-APs against the human colon tumor cell line LoVo uncovered for most of the compounds a cytotoxic potency lower than that of DuP-941 or mitoxantrone and comparable to that of doxorubicin. Only analogues 13c, 13n, and 13ff were as cytotoxic as DuP-941. Interestingly, while DuP-941 was highly cross-resistant in the LoVo cell line resistant to doxorubicin (LoVo/Dx), the 9-aza-APs carrying a distal lipophilic tertiary amine moiety in both chains were capable of overcoming the MDR resistance induced in this cell line. The 9-aza-APs show outstanding in vivo antitumor activity against both systemic P388 murine leukemia and MX-1 human mammary carcinoma transplanted in nude mice. At their optimal dosages, congeners 13a-c, 13f, 13n, 13q, 13x, and 13dd were highly effective against P388 leukemia with T/C% of 200-381, while the T/C% value of DuP-941 was 147. In the MX-1 tumor model, 24 compounds elicited percentages of tumor weight inhibitions (TWI) ranging from 50% to 99%. Congeners 13d, 13k, 13l, 13x, 13z, and 13ee emerged as the most effective ones, with TWI% 96, simliar to that of DuP-941 (TWI% = 95). On the basis of their efficacy profile in additional experimental tumors and lack of cardiotoxicity in preclinical models, two congeners have surfaced as potential clinical candidates.     

Two reconstructive techniques for flatfoot deformity comparing contact characteristics of the hindfoot joints

The effect of two different methods of reconstruction of flatfoot deformity and the role of the posterior tibial tendon on the contact characteristics of the hindfoot joints were quantified using pressure-sensitive film. Each of 10 cadaver feet was loaded quasi-statically by an axial compressive force to simulate varying loads. First, a specimen was tested intact, then it was tested after sectioning the spring ligament and loading the specimen cyclically to create one type of flatfoot deformity. It was then tested again after reconstructing the deformity. Reconstructions used were the Dillwyn-Evans procedure (bone graft in osteotomy of the calcaneus) or the calcaneocuboid distraction arthrodesis (CCDA). We found that surgically produced flatfoot deformity altered mainly the talonavicular joint, by decreasing its contact area. The Dillwyn-Evans method had less effect on the talonavicular joint (altering 2 of 6 measured parameters) than the CCDA (3 of 6) and more effect on the anteriomedial facet (altering 3 of 6 parameters) than the CCDA (1 of 6). The Dillwyn-Evans method had more effect on the posterior facet (altering 2 of 6 measured parameters) than the CCDA (1 of 6). Function of the posterior tibial tendon had no effect on contact characteristics of the hindfoot joints after either type of reconstruction. These findings are based on measurements using a quasi-statically-loaded foot model at three selected positions, and results may be different with dynamic loading.     

Absence of myofibrillar creatine kinase and diaphragm isometric function during repetitive activation

Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. We compared the isometric contractile and fatigue properties of 1) control CK-sufficient (Ctl), 2) M-CK-deficient (M-CK[-/-]), and 3) combined M-CK/ScCKmit-deficient null mutant (CK[-/-]) diaphragm (Dia) to determine the effect of the absence of M-CK activity on Dia performance in vitro. Baseline contractile properties were comparable across groups except for specific force, which was approximately 16% lower in CK[-/-] Dia compared with M-CK[-/-] and Ctl Dia. During repetitive activation (40 Hz, (1)/(3) duty cycle), force declined in all three groups. This decline was significantly greater in CK[-/-] Dia compared with Ctl and M-CK[-/-] Dia. The pattern of force decline did not differ between M-CK[-/-] and Ctl Dia. We conclude that Dia isometric muscle function is not absolutely dependent on the presence of M-CK, whereas the complete absence of CK acutely impairs isometric force generation during repetitive activation.     

Permeability of blood-eye barrier in patients with primary glaucoma combined with lens opacity

The paper focuses on the history and sources of origin of sanology as science, with its brief characterization being given, relating to such items as definition, object and subject of investigation, methodological approaches and tasks, with special emphasis being placed on the significance of sanology in the elaboration of general theory of medicine.     

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.     

Surgical repair of the congenitally malformed mitral valve in infants and children

BACKGROUND: Mitral valve remodeling techniques were applied to 26 infants and children (mean age, 6.0 years, range, 0.4 to 15.9 years) with various forms of congenital mitral valve disease over a 7-year period. Patients with atrioventricular canal, L-transposition and single ventricle were excluded. Intraoperative transesophageal echocardiography (TEE) was utilized to assess the repair and guide the need for immediate intervention. METHODS: Twenty-one patients had mitral regurgitation: 10 with cleft anterior mitral leaflet, 7 with annular dilatation, 1 with normal leaflets with an obstructing cord, 2 with prolapsed leaflets and elongated cords, and 1 with restricted leaflet motion, normal papillary muscles, and shortened cords. Of the 5 mitral stenosis patients, 3 had supravalvular mitral ring, 1 had midvalvular mitral ring, and 1 had a parachute valve. Three of the mitral stenosis patients had additional stenotic lesions. Concurrent repair of associated lesions was performed in 21 patients (78%). RESULTS: Operative mortality was 3.8% (n = 1). There were no late deaths. Immediate rerepair in 4 patients resulted in improved function. All mitral stenosis patients improved. A total of 20 mitral regurgitation patients (95%) improved; 1 required mitral valve replacement. Mean follow-up is 31 months (range, 2 to 81 months). All patients are in New York Heart Association functional class I or II. CONCLUSIONS: Mitral valve repair can be successfully performed in infants and children with excellent short- and midterm results. Assessment using transesophageal echocardiography can guide the necessity for immediate rerepair to achieve improved function.     

Residency training in child sexual abuse evaluation

Insertin is an actin-binding protein that has been isolated from chicken gizzard smooth muscle that has been shown to be highly homologous to amino acids 962-1292 of tensin [Weigt et al., 1992]. Because of the high homology, we investigated the question whether the mRNAs of insertin and of tensin are derived from the same gene by alternative splicing, whether insertin and tensin are encoded by two different genes, or whether insertin is a proteolytic fragment of tensin. In a Northern blot analysis, mRNA from chicken gizzard was hybridized with oligonucleotides specific for tensin and for the insertin domain of tensin. The tensin-specific oligonucleotide hybridized only with the previously reported 8- and 10-kbp RNAs. However, the insertin domain-specific oligonucleotide hybridized with a 1.2 and a 1.6 kbp RNA in addition to the 8 and 10 kbp RNA. The 1.2- and 1.6-kbp RNA occurred in small amounts, as compared with the 8- and 10-kbp RNA. Southern blot analysis of DNA cleaved by the restriction endonucleases BamH1 and HindIII demonstrated that only one gene for the insertin and tensin exists. Insertin isolated from chicken gizzard smooth muscle was investigated by mass spectrometry. The N-termini of three isolated peptides were found to begin at adjacent amino acids and were likely to be formed from tensin by proteolysis. The results suggest that, for insertin, an mRNA exists that is derived from one gene common for insertin and tensin. However, the insertin-specific mRNA contributes relatively little to expression of insertin domains in cells. Insertin preparations from chicken gizzard contain mainly insertin domains formed from tensin by proteolysis.     

The X-ray structure of a divergent cyclophilin from the nematode parasite Brugia malayi

The following is a review of an emerging topic in the literature which has led to new hypotheses regarding the mechanisms of pathogenesis of the various tissue specific AIDS associated syndromes. The fundamental hypothesis in this review proposes that HIV-1 is able to increase lymphocyte and monocyte localization in tissues where released HIV-1 proteins cause local tissue damage leading to any one of the various AIDS associated syndromes. It is also hypothesized here that syndromes associated with other lymphotrophic viruses result from the ability of these viruses to direct leukocyte extravasation of blood vessel walls and to initiate tissue specific pathogenesis. Further, it is suggested here that new concepts and strategies for delivering gene therapy to specific tissues can be derived from our understanding of the mechanisms through which lymphotrophic viruses localize in specific tissues. HIV-1 infection of lymphocytes and monocytes leads to increased adhesion of these cells to vascular endothelium and extracellular matrix molecules. In addition, HIV-1 infection of various leukocytes leads to increased secretion of extracellular matrix degrading matrix metalloproteinases. Increases in leukocyte adhesion and matrix metalloproteinase secretion are associated with the normal mechanisms through which leukocytes localize in tissues during inflammation. The ability of HIV-1 to activate leukocyte adhesion and matrix metalloproteinase secretion suggests that HIV-1 has evolved a way to take advantage of leukocyte inflammatory mechanisms in order to exit the blood stream and gain access to body tissues. The ability of HIV-1 to use infected cells to localize in various tissues may lead to the establishment of HIV-1 reservoirs in tissues. Such viral reservoirs may cause the various tissue specific AIDS associated syndromes. AIDS patients have been found to have elevated adhesion molecules (integrins, and cell adhesion molecules or CAMs) on their peripheral blood lymphocytes (PBLs). While there is little clinical evidence that the tissue localization of HIV-1 infected leukocytes are the cause of the HIV-1 related syndromes, studies in vitro and with animal models have shown that the HIV-1 gene products Tat, Rev and gp120 are potent neurotoxins. It has also been shown that Tat can contribute to the growth of cells from Kaposi's sarcoma lesions. Further, HIV-1 infected cells have been shown to secrete cytotoxic levels of a variety of growth factors and small molecules. Thus, it is likely that the localization of HIV-1 infected cells in specific tissues could contribute to the HIV-1 associated syndromes such as AIDS dementia, HIV-1 related interstitial lung disease, HIV-1 associated nephropathy, the HIV-1 wasting syndrome and perhaps AIDS associated Kaposi's sarcoma and hyperproliferative skin disorders. This review will examine studies in the literature which demonstrate that HIV-1 infection increases leukocyte adhesion and matrix metalloproteinase secretion. Clinical reports of AIDS patient's leukocyte integrin levels will also be reviewed and evidence that tissue localized HIV-1 infected cells could contribute to a variety of HIV-1 associated syndromes will be presented.