Extent of radial sarcomere coupling revealed in passively stretched cardiac myocytes

We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to delayed-extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.     

Changes in cerebral perfusion pressure in puerperal women with preeclampsia

OBJECTIVE: To determine the variation in the estimated maternal cerebral perfusion and cerebrovascular resistance (the resistance area product) in the puerperium. METHODS: The maternal middle cerebral artery was evaluated by transcranial Doppler ultrasound in ten women 2 days before labor, in 21 women in early labor and at 24 and 48 hours postpartum, and in 6 women at 1 week postpartum. Cerebral blood flow velocities were determined. Women were diagnosed initially with mild preeclampsia. Estimated cerebral perfusion pressure was Vmean/[Vmean - Vdiastolic] [BPmean - BPdiastolic]. Because the diameter of the vessels could not be measured directly, an index of resistance was calculated: the resistance area product = BPmean/velocitymean. We calculated an index of cerebral blood flow to be estimated cerebral perfusion pressure divided by resistance area product. Our study had a power of 80% to detect a 16-cm/second increase in middle cerebral blood flow velocity. RESULTS: Estimated maternal cerebral perfusion was maintained for up to 1 week postpartum. Cerebrovascular resistance did not change in the puerperium. Cerebral blood flow index (+/-standard deviation) was significantly increased at 1 week postpartum compared with early labor levels (28.3 +/-6.9 versus 46.7+/-15.6, respectively) (P < .05). CONCLUSION: Cerebral blood flow 1 week postpartum increased significantly over early labor values. These persistent changes in the cerebral vasculature might put patients at risk for seizures up to 1 week postpartum.     

Standardized ultrasound examination for evaluation of instability of the acromioclavicular joint

Anteroposterior X-ray views of both acromioclavicular (AC) joints with 10-kg weights held in each hand are the generally accepted procedure for diagnosis of Tossy I-III grades of AC joint separation. An analogous diagnosis can be made by standardized ultrasound examination. Ten individuals with Tossy-I, 11 with Tossy-II and 8 with Tossy-III instability were examined both radiographically and by B-mode ultrasound. The degree of AC joint separation was uniformly determined on the basis of a calculated index (AC Index = AC joint width of uninjured side/AC joint width of injured side). The mean AC Index for Tossy-I instability determined by ultrasound was 1.0; mean indices of 0.49 and 0.5 were determined for Tossy-II injury by ultrasound and X-ray, respectively, and of 0.21 and 0.2, respectively, for Tossy-III instability. Statistical analysis showed significant differences between the mean AC indices of all three groups (P < 0.0001). We conclude that the reliability of ultrasound examination of AC joint instability is equal to that of radiographic measurement. Standard X-rays of the shoulder remain mandatory only to exclude fracture. The indication for operative stabilization of the AC joint can be established on the basis of the grade of AC joint instability measured by the side-effect-free and cost-effective method of ultrasound examination (AC Index < 0.3 equivalent to Tossy-III instability).     

The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action

Zinc, a common element of adenylate kinases from Gram-positive bacteria, binds to a structural motif consisting of three or four cysteine residues, Cys-X2-Cys-X16-Cys-X2-Cys/Asp. The enzyme from Paracoccus denitrificans, a Gram-negative bacterium, has structural features much similar to those of adenylate kinases from Gram-positive organisms [Spurgin, P., Tomasselli, A.G., and Schiltz, E. (1989) Eur. J. Biochem., 179, 621-628]. However, adenylate kinase isolated from this bacterium was not reported to bind metal. These findings prompted us to clone the corresponding gene of P. denitrificans, and to characterize the enzyme overproduced in Escherichia coli. The deduced primary structure of adenylate kinase from P. denitrificans revealed two differences from that previously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly. The recombinant enzyme is a dimer which binds either zinc or iron, in a metal/monomer ratio of one. The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsulfonate, released the metal from the protein, confirming that thiols are involved in zinc- or iron-binding. The iron-chelated form of recombinant P. denitrificans adenylate kinase, which is essentially under reduced form, transfers electrons to the oxidized cytochrome c. In conclusion, the absence of metal in the enzyme isolated from P. denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.     

Diagnosis and therapy of chronic polyarthritis

Rheumatoid arthritis (RA) is the most frequent inflammatory joint disease, and it affects about 1% of the population. The onset of arthritis is rarely acute; it is subacute and usually progresses slowly. The clinical picture of RA is variable: mild to very aggressive and destructive courses, sometimes accompanied by organ involvement, leading to severe functional impairment and early disability can be observed. RA is diagnosed according to the ACR criteria published in 1958 and modified in 1988. The appearance of a palpable joint swelling or effusion is obligatory for the clinical diagnosis of arthritis. In RA, typically involvement of the joint of the hands and feet can be seen. Laboratory parameters play an important role as both diagnostic and prognostic tools. Besides clinical features and laboratory parameters, imaging techniques provide another cornerstone in the diagnosis of RA. Until now plain X-rays, which primarily visualize osseous changes, are the most important technique in daily practice, whereas magnetic resonance imaging and ultrasound may provide information about soft tissue changes in an earlier stage of disease. The main differential diagnoses of RA to be considered are the seronegative spondylarthropathies (psoriatic arthritis, arthritides accompanying inflammatory bowel diseases, Reiter's syndrome, and spondylitis ankylosans with peripheral arthritis), Parvovirus-induced arthritis, crystal-induced arthritides and septic arthritis. Early diagnosis and therapeutic intervention seem to be of great prognostic importance. In several independently performed investigations a higher mortality was found in RA patients than in the normal population. Drug therapy of RA consists of nonsteroidal antirheumatic drugs (NSAIDs), corticosteroids and disease-modifying drugs (DMARDs). When the functional and radiological parameters were assessed, the DMARDs were found to have a disease modifying and in rare cases a remission-inducing property. Moreover, tolerance these to drugs is limited. Newer therapeutic trials have employed substances like Tenidap, Leflunomid, bacterial extracts, antibiotics and biological subcomes (e.g., monoclonal antibodies against cytokines, fusion proteins for soluble cytokinereceptors). Some promising results of these investigations need confirmation in larger patient populations, but some new perspectives for a more efficacious treatment of RA can be expected.     

Infection prophylaxis in complex limb trauma through immediate definitive reconstruction via a free tissue transfer

Levels of specific IgE antibodies to three kinds of venom (honeybee, wasp and yellow jacket) were followed up for two years in five male pest control operators (age: 24 to 50 years) with Hymenoptera sting within the latest six months. At the first examination, all subjects had a positive reaction for venom-specific IgE but their levels were varied in the range from class 2 to 5 (0.70 approximately 99.9 UA/ml), and a positive reaction was observed in the case of unoffending Hymenoptera. A subject who was re-stung during the two-year follow-up period showed increase in all of the venom-specific IgE levels. Of four subjects without re-stung during the two years, three subjects had a logarithmic decrease in all the specific IgE levels time-dependently, but one subject with a high total serum IgE level showed an increase and decrease alternately in the specific IgE The total serum IgE level may be a factor which influences the change in the venom-specific IgE level. In the four subjects without a high total serum IgE level, the biological half-life of venom-specific IgE was estimated to be 6 to 12 months based on the decreased level during the follow-up period.     

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Blood component therapy and changing transfusion triggers

Transfusion trigger points and appropriate blood component therapy must be continuously evaluated by the anesthesia team during a surgical procedure. This article examines the composition of homologous blood, the storage of blood products, and transfusion reactions. Additionally, the incidence of transfusion transmitted infections is explored. Finally, a review of the current recommendations for transfusions in the clinical setting is provided.