Contractile properties of skeletal muscle fibre bundles from mice deficient in carbonic anhydrase II

The function of cytosolic carbonic anhydrase (CA) isozyme II is largely unknown in skeletal muscle. Because of this, we compared the in vitro contractile properties of extensor digitorum longus (EDL) and soleus (SOL) fibre bundles from mice deficient in CA II (CAD) to litter mate controls (LM). Twitch rise, 1/2 relaxation time and peak twitch force at 22°C of fibre bundles from CAD EDL [28.4±1.4 ms, 31.2±2.3 ms, 6.2±1.0 Newton/cm² (N/cm²), respectively] and CAD SOL (54.2±7.5 ms, 75.7±13.8 ms, 2.9±0.5 N/cm², respectively) were significantly higher compared to LM EDL (20.5±2.2 ms, 21.9±3.7 ms, 4.5±0.2 N/cm²) and LM SOL (42.8±3.5 ms, 51.4±2.4 ms, 2.1±0.4 N/cm²). However, in acidic Krebs–Henseleit solution, mimicking the pH, PCO₂, and HCO₃ ^– of arterial blood from CAD mice, twitch rise, 1/2 relaxation time, and peak twitch force of fibre bundles from CAD EDL (19.3±0.7 ms, 19.7±2.3 ms, 4.8±0.8 N/cm²) and CAD SOL (41.4±3.6 ms, 51.9±5.5 ms, 2.2±0.7 N/cm²) were not significantly different from LM fibre bundles in normal Krebs–Henseleit solution (EDL: 19.7±1.1 ms, 21.6±0.6 ms, 4.7±0.2 N/cm²; SOL: 42.5±3.1 ms, 51.8±2.6 ms, 1.8±0.3 N/cm²). A higher pH_i during exposure to acidic bathing solution was maintained by CAD EDL (7.37±0.02) and CAD SOL (7.33±0.05) compared to LM EDL (7.28±0.04) and LM SOL (7.22±0.02). This suggests that the skeletal muscle of CAD mice possesses an improved defense of pH_i against elevated pCO₂. In support of this, apparent non-bicarbonate buffer capacity (in mequiv H⁺ (pH unit)^–1 (kg cell H₂O)^–1) as determined by pH microelectrode was markedly increased in CAD EDL (75.7±4.1) and CAD SOL (85.9±3.3) compared to LM EDL (39.3±4.7) and LM SOL (37.5±3.8). Both latter phenomena may be related to the slowed rate of intracellular acidification seen in CAD SOL in comparison with LM SOL upon an increase in PCO₂ of the bath. In conclusion, skeletal muscle from mice deficient in CA II exhibits altered handling of acid–base challenges and shows normal contractile behavior at normal intracellular pH.     

Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia. Correlation between VEGF expression and the FAB category

We studied vascular endothelial growth factor (VEGF) expression in bone marrow sections obtained from 3 healthy donors and 41 patients with acute myeloid leukemia (AML) of various French-American-British (FAB) subtypes by immunohistochemical analysis using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody reacted with myeloid progenitor cells and megakaryocytes but not with erythroid cells or mature granulocytic cells. High levels of VEGF were found in the bone marrow in patients with AML-M1, -M2, -M3, -M4, -M4Eo, and -M5. In these leukemias, the vast majority of myeloblasts (> 90%) expressed VEGF. By contrast, in AML-M0, the percentage of VEGF-positive blasts was lower in most cases (median, 42%), and if at all detectable, these blast cells contained only trace amounts of VEGF. In AML-M3 and -M4Eo, maturing granulocytes failed to express VEGF similar to granulocytes in normal bone marrow. In AML-M6, myeloblasts exhibited VEGF, whereas erythroid cells did not. In AML-M7, blast cells and megakaryocytes were identified as major sources of VEGF. In summary, VEGF expression in the bone marrow is restricted to certain stages of differentiation and maturation of myeloid cells and correlates with the FAB category.     

The entorhinal cortex of the Megachiroptera: a comparative study of Wahlberg’s epauletted fruit bat and the straw-coloured fruit bat

This study describes the organisation of the entorhinal cortex of the Megachiroptera, straw-coloured fruit bat and Wahlberg’s epauletted fruit bat. Using Nissl and Timm stains, parvalbumin and SMI-32 immunohistochemistry, we identified five fields within the medial (MEA) and lateral (LEA) entorhinal areas. MEA fields E _CL and E _C are characterised by a poor differentiation between layers II and III, a distinct layer IV and broad, stratified layers V and VI. LEA fields E _I, E _R and E _L are distinguished by cell clusters in layer II, a clear differentiation between layers II and III, a wide columnar layer III and a broad sublayer Va. Clustering in LEA layer II was more typical of the straw-coloured fruit bat. Timm-staining was most intense in layers Ib and II across all fields and layer III of field E _R. Parvalbumin-like staining varied along a medio-lateral gradient with highest immunoreactivity in layers II and III of MEA and more lateral fields of LEA. Sparse SMI-32-like immunoreactivity was seen only in Wahlberg’s epauletted fruit bat. Of the neurons in MEA layer II, ovoid stellate cells account for ~38%, polygonal stellate cells for ~8%, pyramidal cells for ~18%, oblique pyramidal cells for ~6% and other neurons of variable morphology for ~29%. Differences between bats and other species in cellular make-up and cytoarchitecture of layer II may relate to their three-dimensional habitat. Cytoarchitecture of layer V in conjunction with high encephalisation and structural changes in the hippocampus suggest similarities in efferent hippocampal → entorhinal → cortical interactions between fruit bats and primates.     

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase.     

Evaluation of angiogenesis and vascular endothelial growth factor expression in the bone marrow of patients with aplastic anemia

It is generally appreciated that bone marrow function and growth of myelopoietic cells depends on an intact microvasculature. A pivotal regulator of angiogenesis is vascular endothelial growth factor (VEGF). Here, we describe analysis of VEGF expression and microvessel density in the bone marrow of patients with aplastic anemia by immunohistochemistry. Bone marrow was examined at diagnosis and at the time of hematological remission after immunosuppressive therapy using anti-thymocyte globulin, cyclosporin A, and glucocorticoids or allogeneic stem cell transplantation. At diagnosis, both VEGF expression and microvessel density were found to be significantly lower in aplastic anemia compared to normal bone marrow (aplastic anemia, 1.1 +/- 0.7 events per field, versus controls, 5.9 +/- 3.0 events per field; P < 0.05). In response to successful therapy, VEGF and microvessel density in the bone marrow increased substantially. Serum VEGF levels were also found to be significantly lower at diagnosis in aplastic anemia compared to healthy controls (aplastic anemia, 51 +/- 35 pg/ml versus controls, 444 +/- 220 pg/ml; P < 0.05). VEGF in the serum increased substantially after successful immunosuppressive therapy or stem cell transplantation (P < 0.05). Taken together, these data show that aplastic anemia is associated with reduced angiogenesis and reduced VEGF expression.     

Sensitivity of a modified version of the ARCHITECT Anti-HCV test in detecting samples with immunoblot-confirmed, low-level antibody to hepatitis C virus.

BACKGROUND AND OBJECTIVES: Compliance with current regulations regarding the prevention of hepatitis C virus (HCV) transmission in the blood transfusion setting requires the use of sensitive assays for HCV antibody (anti-HCV) detection, which should, ideally, identify any donor having had prior contact with the virus. Therefore, low-level anti-HCV positive blood units should be detected by the screening assays, even those reflecting a past and resolved infection. To assess the sensitivity of two versions of an automated chemiluminescent microparticle immunoassay (CMIA) for anti-HCV screening (ARCHITECT Anti-HCV), 113 single serum samples containing low levels of anti-HCV, assessed by two immunoblot tests, were selected from 3686 samples received for confirmation of HCV infection by a reference laboratory over a 2-year period. MATERIALS AND METHODS: The panel included 17 samples with HCV RNA detected by the polymerase chain reaction (PCR) and 96 PCR negative samples with either positive or indeterminate (anti-Core and anti-NS3 alone) results by immunoblot. RESULTS: All but 13 specimens (100/113, 88.5%) were detected by the current version of the ARCHITECT Anti-HCV assay and 10 additional samples (110/113, 97.3%) tested positive in a modified version of the test. CONCLUSION: The results showed that the modification introduced in the ARCHITECT Anti-HCV assay achieves a significant sensitivity improvement including samples with low-level anti-HCV which are either PCR positive or negative.     

Endosonography of pararectal lymph nodes

One hundred thirteen patients with carcinoma of the rectum were evaluated for lymph node metastases by endorectal ultrasound. With the use of 7.5 MHz and based on different echo patterns, two main groups of lymph nodes can be differentiated: hypoechoic and hyperechoic lymph nodes. Compared with pathologic findings, hypoechoic lymph nodes represent metastases, whereas hyperechoic lymph nodes are visualized due to unspecific inflammation. Lymph node metastases can be predicted with a sensitivity of 72 percent and inflammatory lymph nodes with a specificity of 83 percent. The physical basis of the differentiation of lymph nodes was assessed in vitro by the determination of ultrasound parameters (speed of sound, acoustic impedance, attenuation, and backscattered amplitude). The attenuation coefficient of benign lymph nodes [2.5 dB/(MHz×cm)] is significantly higher than the mean value of lymph node metastases [1.3 db/(MHz×cm)]. The results demonstrate that involved nodes can principally be differentiated from not involved nodes. Micrometastases, mixed lymph nodes, and changing echo patterns within inflammatory nodes explain the accuracy rate of 78 percent.Supported by a grant from the Deutsche Forschungsgemeinschaft Hi 385/1-1     

Chemotactic peptide-induced acute colitis in rabbits

Bacterial chemotactic peptides from the intestinal lumen could potentially induce inflammation if they reached the mucosa. We tested several peptides chemotactic for different inflammatory cells, as well as a nonchemotactic peptide, bradykinin, for their ability to induce colitis in vivo in rabbits. These peptides were also assessed for their ability to stimulate release of the eicosanoids leukotrienes B4 and C4 and prostaglandin E2 from normal rabbit colons perfused ex vivo. Intracolonic administration of n-formyl-methionyl-leucyl-phenylalanine (chemotactic for neutrophils); its methyl ester (chemotactic for monocytes), and alanyl-glycyl-seryl-glutamic acid (chemotactic for eosinophils) all produced colitis (assessed grossly and histologically) within 4 days. Bradykinin did not induce colitis although it did release prostaglandin E2. n-Formyl-methionyl-leucyl-phenylalanine methyl ester induced the greatest degree of colitis in vivo and released prostaglandin E2 and leukotrienes ex vivo. n-Formyl-methionyl-leucyl-phenylalanine and alanyl-glycyl-seryl-glutamic acid induced comparable degrees of inflammation, but alanyl-glycyl-seryl-glutamic acid produced no eicosanoid release while n-formyl-methionyl-leucyl-phenylalanine released both prostaglandin E2 and leukotriene B4 and leukotriene C4 products from normal ex vivo perfused colons. Thus alanyl-glycyl-seryl-glutamic acid produces colitis independent of proinflammatory eicosanoids while eicosanoid release could contribute to colitis produced by n-formyl-methionyl-leucyl-phenylalanine methyl ester. This experimental model of colitis may reflect one possible etiology of inflammatory bowel disease in humans, when bacterial chemotactic peptides breach mucosal defenses in susceptible individuals.     

Functional and aesthetic treatment outcomes after immediate jaw reconstruction using a fibula flap and dental implants

Purpose

Microvascular fibula flap surgery is a reliable and effective procedure for reconstructing the jaws after tumour surgery. This procedure allows the placement of dental implants after bone consolidation. This study was designed to evaluate the oral, functional, and aesthetic rehabilitation of tumour patients with immediate fibula transfer and dental implants and included assessment of diet, speech, and aesthetics.

Predictability and Inducibility of Detachment of Prostatic Central Gland Tissue after Prostatic Artery Embolization: Post Hoc Analysis of a Randomized Controlled Trial

Purpose

To assess the frequency and potential predictors of prostatic central gland tissue detachment (CGD), an enucleation-like reaction that sporadically occurred in a randomized controlled trial assessing efficacy and safety of prostatic artery embolization (PAE).