Treatment of non-traumatic rotator cuff tears: A randomised controlled trial with one-year clinical results.

We have compared three different methods of treating symptomatic non-traumatic tears of the supraspinatus tendon in patients above 55 years of age. A total of 180 shoulders (173 patients) with supraspinatus tendon tears were randomly allocated into one of three groups (each of 60 shoulders); physiotherapy (group 1), acromioplasty and physiotherapy (group 2) and rotator cuff repair, acromioplasty and physiotherapy (group 3). The Constant score was assessed and followed up by an independent observer pre-operatively and at three, six and twelve months after the intervention. Of these, 167 shoulders were available for assessment at one year (follow-up rate of 92.8%). There were 55 shoulders in group 1 (24 in males and 31 in females, mean age 65 years (55 to 79)), 57 in group 2 (29 male and 28 female, mean age 65 years (55 to 79)) and 55 shoulders in group 3 (26 male and 29 female, mean age 65 years (55 to 81)). There were no between-group differences in the Constant score at final follow-up: 74.1 (sd 14.2), 77.2 (sd 13.0) and 77.9 (sd 12.1) in groups 1, 2 and 3, respectively (p = 0.34). The mean change in the Constant score was 17.0, 17.5, and 19.8, respectively (p = 0.34). These results suggest that at one-year follow-up, operative treatment is no better than conservative treatment with regard to non-traumatic supraspinatus tears, and that conservative treatment should be considered as the primary method of treatment for this condition.

Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability.

Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T(d)=74.4 degrees C whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degrees C, respectively, demonstrating significant contribution of the above Trp residues to the structural stability of HLL. Fluorescence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas the opposite was true for W89m. These changes in steady-state emission were sharp, with midpoints (T(m)) at approx. 70.5, 61.0, and 65.5 degrees C for wt HLL, W89L, and W89m, respectively. Both steady-state and time resolved fluorescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated. However, faster mobilities became evident when the unfolding temperatures (T(m)) were exceeded, and the lipases became less compact as indicated by the increased hydrodynamic radii. Even at high temperatures (up to 85 degrees C) a significant extent of tertiary and secondary structure was revealed by circular dichroism. Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL with temperature. Our results also indicate that the thermal unfolding of these lipases is not a two-state process but involves intermediate states. Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the protein to be trapped in an intermediate, higher energy state. The present data show that the mutations, especially W89L in the lid, contribute significantly to the stability, structure and activity of HLL.

Binding of novel peptide inhibitors of type IV collagenases to phospholipid membranes and use in liposome targeting to tumor cells in vitro.

We have recently described a novel cyclic peptide inhibitor CTTHWGFTLC (CTT) for matrix metalloproteinases (MMP)-2 and MMP-9, also called type IV collagenases or gelatinases (E. Koivunen et al., NAT: BIOTECHNOL:, 17: 768-774, 1999). As indicated by its amino acid composition, CTT is hydrophobic, and its partitioning into phospholipid films could be verified by the monolayer technique. Augmented fluorescence emission anisotropy (from 0.064 to 0.349) and reduced collisional quenching by I(-) of the Trp residue in CTT was evident in the presence of unilamellar phosphatidylcholine/phosphatidylethanolamine liposomes, revealing the association of CTT with the lipid bilayers. Gelatinases are potential targets of therapeutic intervention in cancer, and inhibitors of these enzymes can prevent tumor progression in animal models. CTT enhanced 3- to 4-fold the cellular uptake of liposome-encapsulated water-soluble fluorescent marker, rhodamine B by gelatinase-expressing cells. Gelatinase targeting seems to be essential, as modified peptides that were less potent gelatinase inhibitors were also less efficient in promoting the cellular uptake of liposomes. Augmented killing ( approximately 4-fold) of U937 leukemia and HT1080 sarcoma cells was obtained by the CTT-enhanced delivery of Adriamycin-containing liposomes, compared with control liposomes administered without the peptide. These results suggest a novel type of utility for small gelatinase inhibitors in targeted cancer therapy.

Effects of i-propanol on the structural dynamics of Thermomyces lanuginosa lipase revealed by tryptophan fluorescence.

Influence of isopropanol (iPrOH) on the structural dynamics of Thermomyces lanuginosa lipase (TLL) was studied by steady-state, time-resolved, and stopped-flow fluorescence spectroscopy, monitoring the intrinsic emission of Trp residues. The fluorescence of the four Trps of the wild-type enzyme report on the global changes of the whole lipase molecule. To monitor the conformational changes in the so-called "lid," an alpha-helical surface loop, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichroism (CD) spectra revealed that iPrOH does not cause major alterations in the secondary structures of the wild-type TLL and W89m. With increasing [iPrOH], judged by the ratio of emission intensities at 350 nm and 330 nm, the average microenvironment of the Trps in the wild-type TLL became more hydrophobic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time-resolved fluorescence measurements revealed no major changes to be induced by iPrOH neither in the shorter fluorescence lifetime component (tau(1) = 0.5--1.2 ns) for the wild-type TLL nor in the longer fluorescence lifetime component (tau(2) = 4.8--6.0 ns) in the wild-type TLL and the W89m mutant. Instead, for W89m on increasing iPrOH from 25% to 50% the value for tau(1) increased significantly, from 0.43 to 1.5 ns. The shorter correlation time phi(1) of W89m had a minimum of 0.08 ns in 25% iPrOH. Judged from the residual anisotropy r(infinity) the amplitude of the local motion of Trp89 increased upon increasing [iPrOH] 10%. Stopped-flow fluorescence spectroscopy measurements suggested the lid to open within approximately 2 ms upon transfer of W89m into 25% iPrOH. Steady-state anisotropies and longer correlation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.

ATP induces a conformational change in lipid-bound cytochrome c.

Resonance energy transfer studies using a pyrene-labeled phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol (donor) and the heme (acceptor) of cytochrome c (cyt c) have indicated that ATP causes changes in the conformation of the lipid-bound protein (Rytömaa, M., Mustonen, P., and Kinnunen, P. K. J. (1992) J. Biol. Chem. 267, 22243-22248). Accordingly, after binding cyt c via its so called C-site to neat phosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, further quenching of donor fluorescence is caused by ATP, saturating at 2 mm nucleotide. ATP-induced conformational changes in liposome-associated cyt c could be directly demonstrated by CD in the Soret band region (380-460 nm). The latter data were further supported by time-resolved spectroscopy using the fluorescent cyt c analog with a Zn(2+)-substituted heme moiety. A high affinity ATP-binding site has been demonstrated in cyt c (Craig, D. B., and Wallace, C. J. A. (1993) Protein Sci. 2, 966-976) that is compromised by replacing the invariant Arg(91) to norleucine. Although no major effects on conformation and function of cyt c were concluded due to the modification, a significantly reduced effect by ATP on the lipid-bound [Nle(91)]cyt c was evident, implying that this modulation is mediated via the Arg(91)-containing binding site.

Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers.

Fluorescent derivatives of phosphatidyl inositol (PtdIns)-(4,5)-P2 were synthesized and used to test the effects of the PtdIns-(4, 5)-P2-regulated proteins gelsolin, tau, cofilin, and profilin on labeled PtdIns-(4,5)-P2 that was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7-nitrobenz-2-oxa-1,3-diazole (NBD)- or pyrene-labeled PtdIns-(4,5)-P2 and NBD-PtdIns-(3,4,5)-P3. Cofilin and profilin produced no detectable change at equimolar ratios to PtdIns-(4,5)-P2, while tau decreased NBD-PtdIns-(4,5)-P2 fluorescence. Fluorescence enhancement by gelsolin of NBD-PtdIns-(4, 5)-P2 in mixed lipid vesicles depended on the mole fraction of PtdIns-(4,5)-P2 in the bilayer. Specific enhancement of 3% NBD-PtdIns-(4,5)-P2 : 97% PtdCho was much lower than that of 10% PtdIns-(4,5)-P2 : 90% PtdCho, but the enhancement of 3% NBD-PtdIns-(4,5)-P2 could be increased by addition of 7% unlabeled PtdIns-(4,5)-P2. The gelsolin-dependent increase in NBD-PtdIns-(4, 5)-P2 fluorescence was reversed by addition of Ca2+ or G-actin. Significant, but weaker, fluorescence enhancement was observed with the gelsolin N-terminal domain (residues 1-160) and a peptide comprised of gelsolin residues 150-169. Fluorescence energy transfer from gelsolin to pyrene-PtdIns-(4,5)-P2 was much stronger with intact gelsolin than the N-terminal region of gelsolin containing the PtdIns-(4,5)-P2 binding sites, suggesting that PtdIns-(4,5)-P2 may bind near a site formed by the juxtaposition of the N- and C-terminal domains of gelsolin.

The invariant ARG91 is required for the rupture of liposomes by cytochrome C.

Lipid binding properties of cytochrome c (cyt c) were investigated by using semisynthetic mutant protein having amino acid substitution on the evolutionary invariant residue Arg91. We demonstrate here that the membrane binding properties of cyt c are dramatically altered by substituting norleucine for the invariant Arg91. More specifically, while the binding of this mutant to liposomes per se is indistinguishable from the wild type protein, it completely lacks the ability of the native cyt c to rupture liposome bilayers.