Caspase-dependent cleavage of carbamoyl phosphate synthetase II during apoptosis

Carbamoyl phosphate synthetase II (CPSII) is part of carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Herein, we identify CAD as a substrate for caspase-3 degradation in both in vitro and in vivo models of apoptosis. Withdrawal of interleukin-3 or incubation with staurosporine (STS) or doxorubicin (Dox) resulted in proteolytic cleavage of CAD in a myeloid precursor cell line (32D) or in a cell line over-expressing CAD. The rapid decline in the CPSII activity paralleled the degradation of CAD and preceded the appearance of Annexin-V-stained apoptotic cells and DNA fragmentation. These events correlated closely with the activation of caspase-3 in these cells and were prevented by the cell-permeable caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone. Moreover, the incubation of purified CAD with recombinant caspase-3 in vitro generated CAD fragments that were similar to those obtained in vivo. Edman sequencing revealed that two of the major caspase-3 cleavage sites occurred at the sequences EAVD/G and VACD/G within the catalytic (B2) and allosteric (B3) domains of CAD, thus providing a potential mechanism for the rapid inactivation of CPSII during apoptosis. Consistent with this, an enhanced loss of the intracellular pyrimidines (UTP and CTP) was observed in response to STS or DOX-induced apoptosis. Therefore, these studies show that CAD is a novel target for caspase-dependent regulation during apoptosis and suggest that the selective inactivation of pyrimidine nucleotide synthesis accompanies the process of apoptosis.     

Behavioral medicine in the prevention and treatment of cardiovascular disease

Cardiac behavioral medicine is the application of behavioral and psychosocial principles to the prevention and treatment of heart disease. Most biomedical cardiovascular risk factors (e.g., high blood lipids, high blood pressure, diabetes) require behavioral and medical interventions. Other risks, including obesity, high-fat eating pattern, smoking, and inactivity, clearly require lifestyle change. Behavioral medicine screening and intervention have been applied to psychosocial risk factors such as depression, hostility, and social isolation. Appropriate assessment of risk factors is essential because research has demonstrated successful prevention of heart disease and reduction of morbidity and mortality in patients with existing disease. Behavioral interventions have been beneficial in improving cardiac outcomes by enhancing compliance with medication taking and dietary/exercise recommendations. Future needs include the study of psychosocial factors in women and ethnic minorities with heart disease and the integration of behavioral medicine with newer medical technologies designed to detect subclinical biomarkers of heart disease.     

Developmentally regulated monocyte recruitment and bone resorption are modulated by functional deletion of the monocytic chemoattractant protein-1 gene

Tooth eruption involves the movement of a tooth from its site of development within the alveolar bone to its functional position in the oral cavity. Because this process is dependent upon monocytes and formation of osteoclasts, it represents an excellent model for examination of these processes under developmental regulation. We investigated the functional role of monocyte chemoattractant protein-1 (MCP-1) in monocyte recruitment and its impact on bone resorption by examining each parameter in MCP-1(-/-) mice as compared with wild-type controls during tooth eruption. The peak number of monocytes occurred on day 5 in the MCP-1(-/-) mice and on day 9 in the wild-type mice. The peak number of osteoclasts followed the same pattern, occurring sooner in the MCP-1(-/-) (day 5) than in wild-type mice (day 9). Consistent with this, MCP-1(-/-) mice had an accelerated rate of tooth eruption in the early phase when the teeth first entered the oral cavity as compared with the wild-type mice. However, there was accelerated eruption in the wild-type group in the later phase of tooth eruption. When examined at the molecular level, inducible nitric oxide synthase (iNOS) and interleukin-11 and -6 were expressed at considerably higher levels in the experimental group with accelerated tooth eruption. This is the first report identifying these factors as potential modulators of bone resorption that can accelerate the rate of tooth eruption. We conclude that, at early timepoints, monocyte recruitment occurs by MCP-1-independent mechanisms. However, at a later timepoint, MCP-1 may play a contributory role in the recruitment of monocytic cells, allowing the wild-type animals to catch up.     

Update on the prevention and treatment of sexually transmitted diseases

The Centers for Disease Control and Prevention updated its guidelines for the treatment of sexually transmitted diseases. The guidelines include the following information: recommendations for hepatitis A immunization and expanded indications for hepatitis B vaccination; updated diagnostic criteria for pelvic inflammatory disease and parenteral treatment regimens; information on two additional antiviral agents for the treatment of genital herpes; a recommendation for use of a single 1-g dose of azithromycin (Zithromax) to treat urethritis and chlamydial cervicitis; information on the use of quinolones in the treatment of gonococcal infections; information on podofilox and imiquimod, which are both patient-applied medications, in the treatment of noncervical human papillomavirus infection; updated guidelines for the prevention and detection of congenital syphilis; and information on how to prevent the spread of sexually transmitted diseases by educating patients about the importance of changing their sexual behaviors. To have a significant impact on the current rate of transmission of sexually transmitted diseases, family physicians should develop a plan to integrate the guidelines into their practices.     

Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-alpha

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp- like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.      

Molecular analysis of PKU in Ireland

Classical phenylketonuria (PKU: McKusick No. 261600) is caused by mutations occurring at the phenylalanine hydroxylase (PAH) locus on chromosome 12 and has a prevalence in Ireland of 1 in 4500. We examined 304 independent alleles from 350 patients for the presence of six mutations and have characterized VNTR alleles within the minisatellite region 3' to the PAH gene in patients carrying the most prevalent mutation. R408W was the most common mutation found, with a relative frequency of 42%. All other mutations had relative frequencies of <10%. VNTR analysis showed that the R408W mutation is associated with the VNTR-8 allele in the Irish population, indicating that R408W is associated with RFLP haplotype 1. This differs from that reported from eastern Europe where R408W is associated with RFLP haplotype 2/VNTR-3; an observation which has led several groups to propose a Balto-Slavic origin for this mutation. These results support the hypothesis of a second, independent founding event for the R408W mutation on an RFLP haplotype 1 VNTR-8 chromsome background in the Irish/Celtic population.     

Midwifery triage of first trimester bleeding

Approximately one of five pregnant women will experience bleeding during the first trimester of pregnancy. Of these women, about half will go on to have a spontaneous abortion. Comfort with triaging this common problem assists the midwife in providing quality, cost-effective care without eliminating continuity. This article reviews the causes of early pregnancy bleeding, offers strategies to differentiate between these causes, and identifies emergent from nonemergent presentations. Triage and management strategies for women with an impending or threatened spontaneous abortion are reviewed, exploring the alternative of expectant management.     

Tumour marker measurements in the diagnosis and monitoring of breast cancer

Elevation of established blood tumour markers correlates with the stage of breast cancer. The major role of current blood markers is therefore in the diagnosis and monitoring of metastatic disease. A combination of markers is better than a single marker with the most widely adopted combination being CEA and one MUC1 mucin, commonly detected as either CA15.3 or CA27.29. Tumour marker measurement is now used as a complementary test in the diagnosis of symptomatic metastases. In the monitoring of therapeutic response to both endocrine and cytotoxic therapies in advanced disease, biochemical assessment using blood markers not only correlates with conventional UICC criteria but has a lot of advantages which make it a potentially superior way of assessment. In this regard, CA15.3, CEA and ESR are the best validated combination. Studies are ongoing to evaluate the use of sequential blood tumour marker measurements in the follow-up of patients after treatment for their primary breast cancer, in terms of both early detection and early therapeutic intervention. Further randomized studies are also required to ascertain that marker-directed therapy is superior to the current practice for metastatic disease. In line with clinical studies, intensive laboratory work is being carried out to optimize the use of blood markers in advanced disease as well as to exploit their use in screening and diagnosis of early primary breast cancer.