Morphometric evaluation of the linea alba in fresh corpses.

Purpose: Diastasis recti abdominis is an increase in the distance between the medial borders of the two rectus muscles. It is most often triggered after intra-abdominal pressure increases, such as postpartum or in obesity. Most publications are based on radiological studies or are done in certain subgroups, without unanimous reference values of the distance between the rectus abdominis or standardization. Methods: Forty-one cadavers were studied. Exclusion criteria: signs of abdominal trauma, major burns, presence of scar from previous abdominal surgery, clinical signs of abdominal hernia, and identification of hernia during cadaver dissection. Linea alba (LA) length, width, and thickness were measured with a flexible tape measure and digital caliper. Anatomical landmarks were established, and subdivisions were described based on them to compare the cadavers. Results: Sex and age had little effect on LA width, thickness, or length. Obesity (compared to normal weight) was the only variable that promoted an increase in the LA width (p < 0.01). The supraumbilical length varied with the total height of the evaluated cadavers (p < 0.01), but the infraumbilical length did not (p = 0.11). Conclusion: The general statistical results of this study, regarding the evaluation of LA measurements in cadavers, showed that ethnicity, sex, and age have little effect on the width, thickness, or length of the LA. LA width differed significantly with abdominal circumference.

Anti-ophidian activity of Bredemeyera floribunda Willd. (Polygalaceae) root extract on the local effects induced by Bothrops jararacussu venom

Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3′,4′-OH flavonoids are known inhibitors of phospholipases A2.

Anti-ophidian activity of Bredemeyera floribunda Willd. (Polygalaceae) root extract on the local effects induced by Bothrops jararacussu venom.

Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3',4'-OH flavonoids are known inhibitors of phospholipases A2.

In vitro basal and metabolism-mediated cytotoxicity of flavonoids.

The purpose of this study was to compare the basal cytotoxicity and metabolism-mediated cytotoxicity of kaempferol, quercetin and rutin. McCoy cells were exposed to various concentrations of the flavonols with and without the S9 system. The neutral red uptake assay was used to determine viability after 24 h at 35-37 degrees C. Dose-response curves were established for each flavonol in the presence and absence of external metabolizing systems. Kaempferol and quercetin were cytotoxic and provoked a dose-dependent decrease in cell viability, without the S9 system. The hepatic S9 microsomal fraction metabolized these compounds to less cytotoxic metabolites. In contrast, rutin at 500 microg/ml failed to produce any overt signs of toxicity in either assay.

Subunit dissociation and inactivation of pyruvate kinase by hydrostatic pressure oxidation of sulfhydryl groups and ligand effects on enzyme stability.

The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size-exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure-induced subunit dissociation. Size-exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure-dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far-UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K+, Mg2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer.

The absence of p27Kip1, an inhibitor of G1 cyclin-dependent kinases, uncouples differentiation and growth arrest during the granulosa->luteal transition.

The involvement of cyclin-dependent kinase inhibitors in differentiation remains unclear: are the roles of cyclin-dependent kinase inhibitors restricted to cell cycle arrest; or also required for completion of the differentiation program; or both? Here, we report that differentiation of luteal cells can be uncoupled from growth arrest in p27-deficient mice. In these mice, female-specific infertility correlates with a failure of embryos to implant at embryonic day 4.5. We show by ovarian transplant and hormone reconstitution experiments that failure to regulate luteal cell estradiol is one physiological mechanism for infertility in these mice. This failure is not due to a failure of p27-deficient granulosa cells to differentiate after hormonal stimulation; P450scc, a marker for luteal progesterone biosynthesis, is expressed and granulosa cell-specific cyclin D2 expression is reduced. However, unlike their wild-type counterparts, p27-deficient luteal cells continue to proliferate for up to 3.5 days after hormonal stimulation. By day 5.5, however, these cells withdraw from the cell cycle, suggesting that p27 plays a role in the early events regulating withdrawal of cells from the cell cycle. We have further shown that in the absence of this timely withdrawal, estradiol regulation is perturbed, explaining in part how fertility is compromised at the level of implantation. These data support the interpretation of our previous observations on oligodendrocyte differentiation about a role for p27 in establishing the nonproliferative state, which in some cases (oligodendrocytes) is required for differentiation, whereas in other cases it is required for the proper functioning of a differentiated cell (luteal cell).

High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice.

The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators which function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. We report here that nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. We further show that disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life.

Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo.

Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 (Ku70(-/-)). Like Ku80(-/-) mice, Ku70(-/-) mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80(-/-) mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8- and CD4-CD8+ T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.

Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2.

Metanephric mesenchyme gives rise to both the epithelial cells of the nephron and the stromal cells of the mature kidney. The function of the stroma. in kidney morphogenesis is poorly understood. We have generated mice with a null mutation in the Winged Helix (WH) transcription factor BF-2 to examine its function during development. BF-2 expression within the developing kidney is restricted to the stromal cell lineage. Homozygotes die within the first 24 hr after birth with abnormal kidneys. Mutant kidneys are small, fused longitudinally, and rotated 90 degrees ventrally. Histological examination reveals a smaller collecting system, numerous large condensations of mesenchyme, and a decrease in the number of nephrons. Using molecular markers we show that induction and condensation of the nephrogenic mesenchyme occurs normally in mutant. The disruption of BF-2 reduces the rate of differentiation of the condensed mesenchyme into tubular epithelium, as well as the rate of growth and branching of the ureter and collecting system. Our findings demonstrate that BF-2 and stromal cells have essential functions during kidney morphogenesis. Furthermore, they suggest that BF-2 controls the production, by the stroma, of signals or factors that are required for the normal transition of induced mesenchyme into tubular epithelium and full growth and branching of the collecting system.

Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1).

SUMMARY: Disruption of the cyclin-dependent kinase-inhibitory domain of p27 enhances growth of mice. Growth is attributed to an increase in cell number, due to increased cell proliferation, most obviously in tissues that ordinarily express p27 at the highest levels. Disruption of p27 function leads to nodular hyperplasia in the intermediate lobe of the pituitary. However, increased growth occurs without an increase in the amounts of either growth hormone or IGF-I. In addition, female mice were infertile. Luteal cell differentiation is impaired, and a disordered estrus cycle is detected. These results reflect a disturbance of the hypothalamic-pituitary-ovarian axis. The phenotypes of these mice suggest that loss of p27 causes an alteration in cell proliferation that can lead to specific endocrine dysfunction.

Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres.

We generated mice with a null mutation of the forebrain-restricted transcription factor BF-1 to examine its function in brain development. Heterozygous animals have an apparently normal phenotype. Homozygous null BF-1 mutants die at birth and have a dramatic reduction in the size of the cerebral hemispheres. The development of the ventral telencephalon is more severely affected than that of the dorsal telencephalon. Telencephalic neuroepithelial cells are specified in the BF-1 mutant, but their proliferation is reduced. Dorsal telencephalic neuroepithelial cells also differentiate prematurely, leading to early depletion of the progenitor population. These results suggest that BF-1 controls the morphogenesis of the telencephalon by regulating the rate of neuroepithelial cell proliferation and the timing of neuronal differentiation.