Faculty Bibliography

Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to interactions between their respective transmembrane domains. In spite of numerous mutagenesis and kinetic studies, we still do not have a clear mechanistic picture of how PLB influences the calcium transport cycle of SERCA. Herein, we have created alanine mutants for each residue in the transmembrane domain of PLB, we have co-reconstituted these mutants with SERCA into proteoliposomes, and we have performed kinetic simulations of the calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a variable effect on the calcium affinity, maximal activity, and cooperativity of SERCA, such that a range of values was observed. Kinetic simulations using a well-established reaction scheme for SERCA then allowed us to correlate the effects on SERCA activity with changes in the reaction scheme rate constants. Only three steps in the reaction scheme were affected by the presence of PLB, namely, binding of the first calcium ion, a subsequent conformational change in SERCA, and binding of the second calcium ion. The ability of wild-type and mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated with a decreased rate of binding of the second calcium ion. In addition, the ability of wild-type and mutant forms of PLB to alter the maximal activity of SERCA correlated with a change in the forward rate constant for the slow conformational change in SERCA following binding of the first calcium ion.

Mutations in the rhodopsin gene that disrupt the encoded protein's folding properties are a major cause of autosomal dominant retinitis pigmentosa (ADRP). This disease is faithfully modeled in Drosophila where similar mutations in the ninaE gene, encoding rhodopsin-1 (Rh-1), cause ER stress and dominantly trigger age-related retinal degeneration. In addition, mutant flies bearing certain ninaE alleles have dramatically reduced Rh-1 protein levels, but the underlying mechanism for this reduction and significance of its contribution to the ADRP phenotype remains unclear. To address this question, we specifically analyzed the role of Drosophila genes homologous to the known yeast and animal regulators of the ER-associated degradation (ERAD) pathway, a process that reduces levels of misfolded proteins in the ER through proteasomal degradation. We found that loss-of-function of these putative ERAD factors resulted in increased levels of Rh-1 in ninaE mutant flies. Conversely, in an ER stress assay where mutant or wild-type Rh-1 were overexpressed in developing imaginal discs beyond the ER protein folding capacity of those cells, co-expression of certain ERAD factors was sufficient to reduce Rh-1 protein levels and to completely suppress ER stress reporter activation. Significantly, those ERAD factors that specifically reduced misfolded Rh-1 in the imaginal disc assay also delayed age-related retinal degeneration caused by an endogenous ninaE allele, indicating that ERAD acts as a protective mechanism against retinal degeneration in the Drosophila model for ADRP. These results suggest that manipulation of ERAD may serve as a powerful therapeutic strategy against a number of diseases associated with ER stress

The anterior cingulate cortex (ACC) is a component of the limbic system implicated in a wide variety of functions spanning motor and sensory information processing, memory, attention, novelty detection, and comparisons of expectation versus outcome. It remains unclear how much of this functional diversity stems from differences in methodology or interpretation versus truly reflecting the range of processes in which the ACC is involved. In the present study, ACC neuronal activity was examined in freely behaving mice (C57BL6/J) under conditions allowing investigation of many of the cited functions in conditions free from externally applied rules: tests of novel object and novel location recognition memory. Behavioral activity and neuronal activity were recorded first in the open field, during the initial exposure and subsequent familiarization to two identical objects, and finally during the recognition memory tests. No discernible stable firing correlates of ACC neurons were found in the open field, but the addition of objects led to lasting changes in the firing patterns of many ACC neurons around one or both of the object locations. During the novel location test, some neurons followed the familiar object to its new location, others fired exclusively where the object had been, and yet others fired to both current and former object locations. Many of these same features were observed during tests of object recognition memory. However, the magnitude of the neuronal preference for the novel or the familiar object was markedly greater than that observed during either the tests of location recognition or novel object preferences in animals that did not exhibit the expected behavior. The present study reveals, for the first time, single-neuron correlates of object and location recognition behaviors in the rodent ACC and suggests that neurons of the ACC provide a distributed representation of all of the salient features of a task.

OBJECTIVES: To better categorize the epidemiologic profile, clinical features, and disease associations of loose anagen hair syndrome (LAHS) compared with other forms of childhood alopecia. DESIGN: Retrospective survey. SETTING: Academic pediatric dermatology practice. Patients Three hundred seventy-four patients with alopecia referred from July 1, 1997, to June 31, 2007. MAIN OUTCOME MEASURES: Epidemiologic data for all forms of alopecia were ascertained, such as sex, age at onset, age at the time of evaluation, and clinical diagnosis. Patients with LAHS were further studied by the recording of family history, disease associations, hair-pull test or biopsy results, hair color, laboratory test result abnormalities, initial treatment, and involvement of eyelashes, eyebrows, and nails. RESULTS: Approximately 10% of all children with alopecia had LAHS. The mean age (95% confidence interval) at onset differed between patients with LAHS (2.8 [1.2-4.3] years) vs patients without LAHS (7.1 [6.6-7.7] years) (P < .001), with 3 years being the most common age at onset for patients with LAHS. All but 1 of 37 patients with LAHS were female. The most common symptom reported was thin, sparse hair. Family histories were significant for LAHS (n = 1) and for alopecia areata (n = 3). In 32 of 33 patients, trichograms showed typical loose anagen hairs. Two children had underlying genetic syndromes. No associated laboratory test result abnormalities were noted among patients who underwent testing. CONCLUSIONS: Loose anagen hair syndrome is a common nonscarring alopecia in young girls with a history of sparse or fine hair. Before ordering extensive blood testing in young girls with diffusely thin hair, it is important to perform a hair-pull test, as a trichogram can be instrumental in the confirmation of a diagnosis of LAHS

Considerable progress has recently been made in understanding the role of cardiolipin in mitochondria. In this brief review, we discuss new data that show how cardiolipin specifically contributes to the lateral organization of mitochondrial membranes. We argue that the function of cardiolipin has to be understood in the context of dynamic membrane assembly rather than static membrane structure, and we propose that remodeling of cardiolipin, i.e. the formation of uniformly substituted molecular species, may reduce the energy barrier of the assembly process

The lifetime risks for both breast and ovarian cancer for BRCA mutation carriers far exceeds the general population risk of 13% for breast cancer and 1.4% for ovarian cancer. BRCA carriers have unique and medically complicated decisions to make regarding their cancer treatment or risk reduction. As BRCA testing becomes increasingly common among unaffected individuals in families with a previously documented BRCA mutation, there are a growing number of individuals with unique psychosocial needs and concerns. This review paper describes the BRCA 1/2 population, discusses preimplantation genetic diagnosis (PGD), and describes the decisions and ethical issues related to PGD among the BRCA 1/ 2 population.

OBJECTIVE: To determine the effects of smoking on eggs and subsequent embryo development by maternal exposure to cigarette smoke. DESIGN: Mice were exposed to cigarette smoke or cigarette smoke condensate (CSC) for 4 weeks and then examined for development and telomere function of embryos in vitro after fertilization. In addition, the effects of continuous smoke on embryo development and telomere length were determined by treating mice for 4 weeks, followed by continous exposure to cigarette smoke or CSC after fertilization. SETTING: Laboratory study. ANIMAL(S): CD1 mice. INTERVENTION(S): Mice were exposured to cigarette smoke or CSC. MAIN OUTCOME MEASURE(S): The percentage (rate) of blastocyst development, quality of embryos assessed by total cell number, apoptosis, Oct4 expression (a molecular marker of embryonic stem cells), telomere length and loss, and chromosomal instability were compared between smoke- and CSC- treated mice and sham-treated mice. RESULT(S): Mice exposed to cigarette smoke or CSC for 4 weeks exhibited increased egg fragmentation or delayed fertilization, thus reducing development to blastocysts in vitro. Fragmented eggs showed increased reactive oxygen species. Mice exposed to smoke or CSC showed increased apoptosis and altered expression of Oct4 in developed embryos. The effects of smoke or CSC on embryo development showed a dose-dependent relationship to exposure time. Exposure to smoke or CSC beginning 4 weeks before fertilzation altered expression of Oct4 and increased apoptosis in blastocysts. Notably, the rate of abnormal embryos significantly increased in the smoke and CSC groups. Smoke and CSC shortened telomeres in embryos, but their telomere shortening was not enough to induce major chromosome abnormalities in mice, which have unusually long telomeres. CONCLUSION(S): Together, the whole animal exposure model shows that cigarette smoke induces oxidative stress, telomere shortening, and apoptosis, and compromises embryo development in vivo

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimer's disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine-dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Abeta(1-42) levels by about three fold, beta-CTF (C99) levels by 33% and beta-secretase (BACE1) protein levels by 43%. TD-induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Abeta and promotes accumulation of plaques independent of neuron loss or neuritic clusters

Despite their rarity, diseases of premature aging, or 'progeroid' syndromes, have provided important insights into basic mechanisms that may underlie cancer and normal aging. In this review, we highlight these recent developments in Hutchinson-Gilford progeria syndrome (HGPS), Werner syndrome, Bloom syndrome, Cockayne syndrome, trichothiodystrophy, ataxia-telangiectasia, Rothmund-Thomson syndrome, and xeroderma pigmentosum. Though they are caused by different mutations in various genes and often result in quite disparate phenotypes, deciphering the molecular bases of these conditions has served to highlight their underlying basic similarities. Studies of progeroid syndromes, particularly HGPS, the most dramatic form of premature aging, have contributed to our knowledge of fundamental processes of importance to skin biology, including DNA transcription, replication, and repair, genome instability, cellular senescence, and stem-cell differentiation