Biomedical subjects
Publications and source records attributed to L Shapiro.
The genetic mechanisms that control asymmetric cell divisions--yielding progeny cells that differ from one another--have been conserved among prokaryotes, eukaryotic microbes, and higher organisms. All use the paradigm of regulatory protein localization as a way of translating genetic information into three-dimensional space.
The complex functions of the nervous system reflect the coordinated action of intricately connected networks of neurons. Recent work has combined to suggest the importance of the cadherin family of cell adhesion molecules in specifying these connections at neural synapses. High-resolution structural studies and site-directed mutagenesis have revealed some of the molecular mechanisms by which cadherins function in cell-to-cell adhesion, and suggest new avenues for investigation.
New research on bacterial cells has demonstrated that they have a dynamic and complex subcellular organization. Work in Caulobacter crescentus shows that essential and nonessential proteins localize to discrete positions in the cell as a function of cell-cycle progression. The flagellum and chemotaxis receptor are asymmetrically localized to a single pole in the predivisional cell by coordinated proteolysis and transcriptional regulation. Cell type- and compartment-specific localization of the CtrA global transcriptional regulator is essential for proper cell-cycle progression, and subcellular localization of key chromosome partitioning proteins is correlated with proper nucleoid segregation. Given this structural complexity, we are driven to ask how localization is achieved, and to what end.
The present study was to determine whether the administration of a single dose of interferon-alpha2B (IFN-alpha2B) to healthy humans affects endogenous (or basal level) or inducible cytokines in a whole blood, ex vivo culture. Twenty-four healthy volunteers received an s.c. injection of IFN-alpha2b (3 x 10(6)U), and 4 volunteers received the vehicle as placebo. The study was blinded. Blood was drawn before and 3, 6, 12, and 24 h after the injection and incubated in the presence or absence of lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta). After 24 hs, the plasma was assayed for tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, IL-1beta, IL-1 receptor antagonist (IL-1Ra), and IL-8. Treatment with IFN-alpha2b was associated with a 4.8-fold increase in the endogenous production of IL-1Ra in cultured blood sustained over 24 hs. In contrast, no change in endogenous IL-1Ra production was detected in the controls. A significant suppression (75%, p < 0.001) of IL-1beta-induced IL-8 production 3 and 6 h after IFN-alpha2b compared with control subjects was observed. These effects were also observed when IFN-alpha2b was added directly to whole blood cultures in vitro. In contrast to IL-1 stimulation, LPS stimulation of blood from IFN-alpha2b-treated subjects resulted in enhanced IL-1beta and TNF-alpha production. These results suggest that a single dose of IFN-alpha2b induces an anti-inflammatory state for endogenous stimuli but a proinflammatory state for exogenous endotoxin.
The ordered assembly of the Caulobacter crescentus flagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarchy of four classes. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene, fliX. A fliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Epistasis experiments placed fliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. The fliX gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. Full expression of fliX was found to be dependent on ctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter. The fliX gene is located upstream and is divergently transcribed from the class III flagellar gene flgI, which encodes the basal body P-ring monomer. Analysis of the fliX-flgI intergenic region revealed an arrangement of cis-acting elements similar to that of another set of Caulobacter class II and class III flagellar genes, fliL-flgF, that is also divergently transcribed. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.
To assess the tumor targeting, safety, and efficacy of monoclonal antibody 131I-labeled CC49 in patients with androgen-independent prostate cancer, 16 patients received 75 mCi/m2 of the radiolabeled antibody after 7 days of IFN-gamma pretreatment. Sequential tumor biopsies in three patients showed a median 5-fold (range, 2-6-fold) increase in the proportion of cells staining positively for the TAG-72 antigen, whereas one showed a decrease in staining. Fourteen patients received 131I-labeled CC49, whereas 2 showed a disease-related decrease in performance status, precluding antibody treatment. The antibody localized to sites of metastatic androgen-independent prostate cancer in 86% (12 of 14; 95% confidence interval, 57-95%) of cases. Both osseous and extraosseous sites were visualized, and in six (42%) patients, more areas were visible when the radioimmunoconjugate was used than were apparent when conventional scanning techniques were used. The localization of the conjugate in the marrow cavity was usually a site not visualized by the radionuclide bone scan, in which the isotope localizes primarily to the tumor-bone interface. The dose-limiting toxicity was thrombocytopenia because five (36%) patients showed grade IV and seven (50%) showed grade III effects. In addition, six (42%) patients, four of whom were hospitalized, showed a flare in baseline pain, and four showed a decrease in pain. No patient showed a >50% decline in prostate-specific antigen, although radionuclide bone scans remained stable in four cases for a median of 4 months. The results are consistent with dosimetry estimates showing that the delivered dose to tumor was subtherapeutic and suggest that approaches that exclusively target the bone tumor interface or the marrow stroma may be unable to completely eradicate disease in the marrow cavity. For CC49, improving outcomes would require repetitive dosing, which was precluded by the rapid development of a human antimouse antibody response.
QUESTIONOne of my patients conceived while using a topical tretinoin preparation for acne. I know this drug is related to Accutane, which is teratogenic. How should I advise her?ANSWERAvailable evidence suggests that topical tretinoin does not increase teratogenic risk in humans.
Initially described in 1989 as interferon-gamma (IFN-gamma) inducing factor (IGIF), interleukin-18 (IL-18) is a novel pro-inflammatory cytokine that is clearly more than an inducer of IFN-gamma. The cytokine possesses several biological properties such as activation of nuclear factor-kappaB (NF-kappaB), Fas ligand expression, the induction of both CC and CXC chemokines, and increased production of competent human immunodeficiency virus. Most activities are due to a receptor complex that recruits the IL-1 receptor-activating kinase (IRAK), leading to translocation of NF-kappaB. This property and others support the concept that IL-18 is related to the IL-1 family. Indeed, one of the IL-18 receptor chains is the IL-1 receptor-related protein, a member of the IL-1R family. In addition, IL-18 is structurally similar to IL-1beta and like IL-1beta is first synthesized as a leaderless precursor requiring the IL-1beta converting enzyme for cleavage into an active molecule. The biology of IL-18 is reviewed in the overview and the implication for a role for this cytokine in disease is presented.
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beta-catenin plays a central part in cell adhesion as a structural component of the cadherin complex. In a seemingly disparate role, it is also important in embryo patterning, and now has emerged as a leading actor in carcinogenesis. beta-catenin achieves its diverse functions by interacting with many partners. The recent structure of the core domain from beta-catenin suggests how this talented molecule can achieve its many functions.
A temperature-sensitive (ts) mutation in the ffs gene, encoding 4.5 S RNA, gives rise to cell division and DNA replication defects in Caulobacter crescentus. The ffs gene is transcribed throughout the cell-cycle and is transcribed at similar rates in mutant (ffs36) and wild-type strains, but in the mutant the 4.5 S RNA is unstable leading to lower 4.5 S RNA levels. The ffs36 phenotype results from a single base change in one of the non-conserved stems of the mature RNA, and is completely rescued by a compensating mutation in the opposite strand, providing confirmation of the predicted secondary structure of the 4.5 S RNA. The Caulobacter ffs gene was shown to be functionally comparable to the Escherichia coli ffs gene by complementation. Comparison of the ffs36 strain to a ts secA strain of Caulobacter, also having cell-cycle and DNA replication phenotypes, showed that both exhibit a permanent induction of a heat shock response at the restrictive temperature. To explain the phenotype of both the secA and ffs36 strains, we propose that a cell-cycle checkpoint prevents further progression through the cell-cycle in response to increased intracellular levels of heat shock and misfolded proteins.
The global transcriptional regulator CtrA controls multiple events in the Caulobacter cell cycle, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems and is activated by phosphorylation. We report here that this phosphorylation signal enters the cell cycle at mid S phase. In addition, CtrA function is modulated by temporally and spatially controlled proteolysis. When an active CtrA protein is present at the wrong time in the cell cycle, owing to expression of a mutant CtrA derivative that is active in the absence of phosphorylation and is not turned over during the cell cycle, the G1-to-S transition is blocked and the cell cycle aborts. Thus, both phosphorylation and proteolysis are critical determinants of bacterial cell cycle control in a manner that is analogous to the control of the eukaryotic cell cycle.
A major breakthrough in understanding the bacterial cell is the discovery that the cell is highly organized at the level of protein localization. Proteins are positioned at particular sites in bacteria, including the cell pole, the incipient division plane, and the septum. Differential protein localization can control DNA replication, chromosome segregation, and cytokinesis and is responsible for generating daughter cells with different fates upon cell division. Recent discoveries have revealed that progression through the cell cycle and communication between cellular compartments are mediated by two-component signal transduction systems and signaling pathways involving transcription factor activation by proteolytic processing. Asymmetric cell division in Caulobacter crescentus and sporulation in Bacillus subtilis are used as paradigms for the control of the cell cycle and cellular morphogenesis in bacterial cells.
The synthesis of proinflammatory cytokines involves members of the mitogen-activated protein (MAP) kinase stress pathway, particularly p38 MAP kinase and c-jun NH2-terminal kinase. In this report we used hyperosmotic stress to study changes in steady-state mRNA levels and synthesis of proinflammatory cytokines in freshly obtained human peripheral blood mononuclear cells (PBMC) in vitro. There was no evidence of interleukin (IL)-8 gene expression in freshly obtained human blood despite 30 cycles of amplification of reverse-transcribed mRNA using the polymerase chain reaction. In contrast, exposure of PBMC to hyperosmotic conditions (330-410 mOsM) by the addition of NaCl to tissue culture medium induced gene expression for IL-1 alpha, IL-1 beta, and IL-8. Routine tissue culture medium is hyperosmotic (305 mOsM) compared to human plasma (280-295 mOsM), but decreasing the osmolarity to the physiological range resulted in a 50% reduction in baseline IL-8 synthesis (P < 0.001). Although hyperosmotically induced accumulation of steady-state mRNA levels for IL-1 alpha and IL-1 beta increased 50- and 7-fold over control, respectively, these were poorly translated into each respective cytokine. However, in PBMC stimulated by hyperosmotic stress, the addition of femtomolar concentrations of bacterial lipopolysaccharide, IL-1, or 1% normal human serum resulted in a synergistic synthesis (at least twice that expected) of IL-1 alpha, IL-1 beta, TNF-alpha, and IL-8.
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This paper describes the preparation and characterization of a three-dimensional, porous sponge made from the marine polysaccharide alginate for creating a cell-matrix transplant to replace damaged organs or tissues. The sponge is prepared by a three-step procedure: first gelation of the alginate with bivalent cations, followed by freezing of the hydrogel and finally lyophilization to produce a porous sponge. The pattern and the extent of sponge porosity, as well as its mechanical properties, were influenced by the concentration and the type of alginate (guluronic to mannuronic ratio and viscosity), the type and concentration of the cross-linkers and the freezing regime. By controlling these variables, macroporous sponges (pore size of 70-300 microns) that are suitable for cell culture and neovascularization were achieved. Fibroblasts seeded within the sponges preferred the pores, where they maintained a spherical shape. The alginate sponges conserved their initial volume for at least 3 months. It appears that alginate sponges may provide an excellent support for cell transplantation.
The heavy use of antibiotics over recent decades has resulted in widespread resistance of bacteria to many drugs. Overcoming resistance requires new approaches to antibiotic development, including the exploitation of new targets in the bacterial cell. Protein secretion is essential for bacterial cell growth and virulence, so it could be a suitable target for new therapeutic agents.
Although serious reactions comprise only a small percentage of total adverse drug reactions, they are important in terms of morbidity and potential mortality. An update on serious dermatologic reactions in children is presented including serum sickness-like reactions due to cefaclor, hypersensitivity syndrome reactions (HSRs), and drug-induced pseudoporphyria. More detailed information on minocycline-induced reactions including drug-induced lupus and HSRs and lamotrigine-induced toxic epidermal necrolysis and Stevens-Johnson syndrome will be discussed.