Randomized trial of custom orthoses and footwear on foot pain and plantar pressure in diabetic peripheral arterial disease.

AIM: Custom-made foot orthoses reduce plantar pressure, ulceration and amputation in patients with diabetes mellitus. There is limited evidence of their effect on foot pain. In a randomized, single-blind, sham-controlled trial, the efficacy of custom orthoses on foot pain and plantar pressure in diabetic patients with peripheral arterial disease was investigated. METHODS: Sixty-one participants were randomly assigned to either custom foot orthoses (n = 30) or sham insoles (n = 31). Both groups also received standardized walking footwear. Outcomes included foot pain and function, mean pressure, toe-brachial index, average daily steps, disability, comfort, quality of life, adherence and adverse events. A multivariate predictive model was constructed to explore factors contributing to pain relief during the trial. RESULTS: At 8 weeks, 95% of participants provided follow-up data, adherence was high and there were few adverse events. Foot pain and function scores significantly improved at 8 weeks with both custom orthoses and the sham, but there was no significant difference between groups. Custom orthoses reduced pressure significantly more than the sham. There were no significant differences between groups for toe-brachial index, daily steps, disability, comfort or quality of life. Regression modelling identified inappropriate pre-trial footwear as the strongest predictor of foot pain relief during the trial. CONCLUSIONS: Custom foot orthoses significantly reduced plantar pressure compared with the sham, but there were no significant differences between groups for pain or function. The high-quality walking footwear provided to both groups may explain this finding. Footwear should have greater emphasis as the clinical intervention.

Biology of the CAPA peptides in insects.

CAPA peptides have been isolated from a broad range of insect species as well as an arachnid, and can be grouped into the periviscerokinin and pyrokinin peptide families. In insects, CAPA peptides are the characteristic and most abundant neuropeptides in the abdominal neurohemal system. In many species, CAPA peptides exert potent myotropic effects on different muscles such as the heart. In others, including blood-sucking insects able to transmit serious diseases, CAPA peptides have strong diuretic or anti-diuretic effects and thus are potentially of medical importance. CAPA peptides undergo cell-type-specific sorting and packaging, and are the first insect neuropeptides shown to be differentially processed. In this review, we discuss the current knowledge on the structure, distribution, receptors and physiological actions of the CAPA peptides.

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action.

In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

Periviscerokinins in cockroaches: release, localization, and taxon-specific action on the hyperneural muscle.

The periviscerokinins of Periplaneta americana (Pea-PVKs) were the first neuropeptides directly isolated from perisympathetic organs (PSOs), neurohemal swellings of the transverse and median nerves of insects. It has been demonstrated that Pea-PVK-1 release can be induced from the abdominal PSOs of P. americana by in vitro depolarization. A myotropic effect of Pea-PVK-1 on the hyperneural muscle is restricted to blattid cockroaches, whereas proctolin induces contractions of this muscle in all cockroach species investigated. The location and morphology of Pea-PVK-1-like immunoreactive neurons in species of different cockroach taxa are very similar to those previously described for P. americana. Pea-PVK-1-like immunoreactivity is restricted to cells of the abdominal ganglia which constitute a neurohemal system and project via the median nerve to the abdominal PSOs. Despite interspecific differences in the topography of the transverse nerves of the terminal ganglion, Pea-PVK-1-like immunoreactive fibers always innervate transversal nerves VII and VIII of the terminal ganglion. The results suggest that PVKs act as neurohormones throughout the cockroaches, although they may have different effect(s) depending on the species.

Peptide-induced Ca(2+) movements in a tonic insect muscle: effects of proctolin and periviscerokinin-2.

Although most of the characterized insect neuropeptides have been detected by their actions on muscle contractions, not much is known about the mechanisms underlying excitation-contraction coupling. Thus we initiated a pharmacological study on the myotropic action of the peptides periviscerokinin-2 (PVK-2) and proctolin on the hyperneural muscle of the cockroach Periplaneta americana. Both peptides required extracellular Ca(2+) to induce muscle contraction, and a blockage of sarcolemmal Ca(2+) channels by Mn(2+) or La(3+) inhibited myotropic effects. The peptides were able to induce contractions in dependence on the extracellular Ca(2+) concentration in muscles depolarized with high K(+) saline. A reduction of extracellular Na(+), K(+), or Cl(-) did not effect peptide action. Nifedipine, an L-type Ca(2+)-channel blocker, partially blocked the response to both peptides but to a much lesser extent than contractions evoked by elevated K(+). Using calcium imaging with fluo-3, we show that proctolin induces an increase of the intracellular Ca(2+) concentration. In calcium-free saline, no increase of the intracellular Ca(2+) concentration could be detected. The inhibiting effect of ryanodine, thapsigargin, and TMB-8 on peptide-induced contractions suggests that Ca(2+) release from the sarcoplasmic reticulum plays a major role during peptide-induced contractions. Preliminary experiments suggest that the peptides do not employ cyclic nucleotides as second messengers, but may activate protein kinase C. Our results indicate that the peptides induce Ca(2+) influx by an activation or modulation of dihydropyridine-sensitive and voltage-independent sarcolemmal Ca(2+) channels. Ca(2+)-induced Ca(2+) release from intracellular stores, but not inositol trisphosphate-induced Ca(2+) release, seems to account for most of the observed increase in intracellular Ca(2+). Additionally, both peptides were able to potentiate glutamate-induced contractions at threshold concentrations.

Spin labeling analysis of structure and dynamics of the Na(+)/proline transporter of Escherichia coli.

With respect to the functional importance attributed to the N-terminal part of the Na(+)/proline transporter of Escherichia coli (PutP), we report here on the structural arrangement and functional dynamics of transmembrane domains (TMs) II and III and the adjoining loop regions. Information on membrane topography was obtained by analyzing the residual mobility of site-specifically-attached nitroxide spin label and by determination of collision frequencies of the nitroxide with oxygen and a polar metal ion complex using electron paramagnetic resonance (EPR) spectroscopy. The studies suggest that amino acids Phe45, Ser50, Ser54, Trp59, and Met62 are part of TM II while Gly39 and Arg40 are located at a membrane-water interface probably forming the cytoplasmic cap of the TM. Also Ala67 and Glu75 are at a membrane-water interface, suggesting a location close to the periplasmic ends of TMs II and III, respectively. Ser71 between these residues is clearly in a water-exposed loop (periplasmic loop 3). Spin labels attached to positions 80, 86, and 91 show EPR properties typical for a TM location (TM III). Leu97 may be part of a structured loop region while Ala107 is clearly located in a water-exposed loop (cytoplasmic loop 4). Finally, spin labels attached to the positions of Asp33 and Leu37 are clearly on the surface of the transporter and are directed into an apolar environment. These findings strongly support the recently proposed 13-helix model of PutP [Jung, H., Rübenhagen, R., Tebbe, S., Leifker, K., Tholema, N., Quick, M., and Schmid, R. (1998) J. Biol. Chem. 273, 26400-26407] and suggest that TMs II and III of the transporter are formed by amino acids Ser41 to Gly66 and Ser76 to Gly95, respectively. In addition to the topology analysis, it is shown that binding of Na(+) and/or proline to the transporter alters the mobility of the nitroxide group at the positions of Leu37 and Phe45. From these findings, it is concluded that binding of the ligands induces conformational alterations of PutP that involve at least parts of TM II and the preceding cytoplasmic loop.

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin.

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.

Lesion development in Marburg's type of acute multiple sclerosis: from inflammation to demyelination.

We report a patient who suffered from acute inflammatory CNS demyelination and underwent two consecutive diagnostic stereotactic brain biopsies during the early disease course. The first lesion was drawn 33 days after the onset of disseminated neurological symptoms. Macrophages and T lymphocytes diffusely infiltrated small vessel walls and the white matter. mRNA for tumor necrosis factor alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) was abundantly expressed. Myelin sheaths were entirely preserved. The second biopsy 76 days later showed confluent demyelinating lesions with a diffuse infiltration of macrophages that were positive for myelin debris, activation markers and TNFalpha and iNOS mRNA. IgG and C9neo deposits were found along myelin sheaths. The patient had received intravenous immunoglobulins (IVIG) prior to biopsy. Findings from this single patient affirm that demyelination follows the migration of inflammatory cells from the circulation into the white matter with subsequent inflammation and demyelination. Inflammation alone may be sufficient to cause significant clinical deficits without demyelination. Inflammatory mediators such as TNFalpha and NO are involved at very early stages in the pathogenetic process. IVIG treatment may lead to the deposition of immunoglobulins and to the activation of the complement cascade, but the clinical relevance of this particular finding remains uncertain.

Quantification of periviscerokinin-1 in the nervous system of the American cockroach, Periplaneta americana. An insect neuropeptide with unusual distribution.

This study was undertaken to reveal the quantitative distribution of the insect neuropeptide periviscerokinin-1 (Pea-PVK-1) in the central nervous system of Periplaneta americana and to demonstrate that neurons stained in a previous immunohistochemical study contain authentic Pea-PVK-1. For this, we combined ELISA, HPLC, and MALDI-TOF mass spectrometry. The high specificity of the used antiserum enabled the quantification of Pea-PVK-1 in unseparated tissue extracts. No cross-reactivities with other insect neuropeptides were detected in ELISA. Only two immunoreactive fractions, coeluting with synthetic Pea-PVK-1 in its oxidized and nonoxidized form, were found in HPLC-separated extracts of the brain, suboesophageal ganglion, metathoracic ganglion, second abdominal ganglion with or without perisympathetic organ, and terminal ganglion. By using MALDI-TOF mass spectrometry, we were able to confirm the existence of authentic Pea-PVK-1 in these fractions. The abdominal perisympathetic organs contained 6.3 pmol Pea-PVK-1 per animal; another 1.3 pmol were found in the abdominal ganglia. More than 90% of the total 8.2 pmol in the central nervous system was found in the abdominal ganglia and their perisympathetic organs. The corpora cardiaca and corpora allata did not contain immunoreactive material, suggesting that Pea-PVK-1 is not released by the cephalic neurohaemal system. The quantitative distribution of Pea-PVK-1 differs considerably from that of other known insect neuropeptides.

Isolation and characterization of alfalfa-nodulating rhizobia present in acidic soils of central argentina and uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809-1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.

Transplanting two unique beta-glucanase catalytic activities into one multienzyme, which forms glucose.

Endo cellulases of plant pathogenic erwinias degrade cellulose as well as the cellulosic domains of barley (1-3,1-4)-beta-glucan. Depolymerization of the latter substrate is mainly caused by (1-3,1-4)-beta-glucanases, which hydrolyze (1-4)-beta glycosidic linkages adjacent to (1-3)-beta linkages. To construct an enzyme for efficient degradation of barley (1-3,1-4)-beta-glucan, the sequence encoding the catalytic domain and interdomain linker of the cellulase from Erwinia carotovora subspecies atroseptica was fused to that for the heat stable Bacillus hybrid, H(A12-M) delta Y13 (1-3,1-4)-beta glucanase. The chimeric enzyme secreted from Escherichia coli cells did not remain covalently assembled as judged by SDS-PAGE. However, the glycosylated and intact enzyme (denoted CELGLU) is secreted from the yeast Pichia pastoris. CELGLU exhibits both cellulase and (1-3,1-4)-beta-glucanase catalytic activities, and was accordingly classified a true multienzyme. HPLC and NMR analyses revealed that among the products from CELGLU, di- and trimeric oligosaccharides were identical to those produced by the parental cellulase. Tetrameric oligosaccharides, derived from the (1-3,1-4)-beta-glucanase activity of CELGLU, were further degraded by the cellulase moiety to yield glucose and trimers. Compared with the parental enzymes, CELGLU exhibits substantially higher Vmax for degradation of both soluble cellulose and barley (1-3,1-4)-beta-glucan. These findings point to construction of multienzymes as an effective approach for engineering enzymes with novel characteristics.

Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin.

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7.5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli-pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31% esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.

[Opinions and expectations of physicians of the Halle district for restructuring health care]. / Meinungen und Erwartungen der Arzte des Bezirkes Halle zur Umgestaltung des Gesundheitswesens.

The article reports on a sociological investigation on physicians' views and expectations regarding the restructuring of the East German health system. 1,420 physicians of a former GDR district (= some 3.5 per cent of GDR doctors) were questioned. The data were ascertained in April 1990 via questionnaire.

[The relevance of sonography in the diagnosis of acute abdomen in the intensive care unit]. / Die Relevanz der Sonographie in der Diagnostik des akuten Abdomens auf der Intensivstation.

This study included 414 unselected sonographic examinations of the abdomen of patients in intensive care complaining of acute abdominal symptoms. Examinations of adequate quality showed a sensitivity of 88.8%, a specificity of 76.3% and an accuracy of 83.7%. Even when allowing for examination which could not be evaluated because of the presence of scars, bandages or air, there was a sensitivity of 72.9% and an accuracy of 74.2%. Sonography is therefore the examination of first choice. In a quarter of all cases, however, computer tomography examination was also necessary.

[Secondary vascular disease in spondylolisthesis]. / Sekundäre Gefässerkrankungen bei Spondylolisthesis.

The topographic relation of the spine to the great vessels let to the expectation that spondylolisthesis may lead to mechanical irritation of the cauda equina as well as of the great vessels. The angiological examination of 223 patients with spondylolisthesis of the lumbar spine shows that the changes in the anatomy of the great vessels are less significant than expected after experiments involving artificial dislocations of the spine in cadavers. In vivo arterial function is not altered even in true spondyloptosis. Pelvic veins on the other hand show considerable compression and diminished flow in third and fourth degree spondylolisthesis of L5 over S1 as well as in second degree spondylolishtesis of L4 over L5. These patients seem to be disposed to venous disorders involving varices and thrombosis. If therefore a high venous pressure is found and a ventral fusion proposed it should be discussed to combine the procedure with a decompression of the veins.