Molecular characterization of a gene required for entry into S phase of the mitotic cell cycle in the yeast {\it Saccharomyces cerevisiae\/}

A strain of Saccaromyces cerevisiae (SC3B) with a temperature sensitive defect in the synthesis of DNA has been isolated. This defect is due to a single recessive mutation in a gene named INS1 required for the initiation of S phase. Arrested cells carrying the ins1$\sp{ts}$ allele are defective in the completion of G1 to S phase transition events including SPB duplication or separation, initiation of DNA synthesis, normal control of budding, and bud neck stability. The mutation and a gene which complements the mutation were mapped to chromosome IV. The complementing gene was proved to be the wild type allele of the temperature sensitive mutation by genetic linkage of an integrated clone. A very low abundance 4.2 kb RNA message was observed in the strain SC3B which increased greatly in this strain transformed with a multiple copy plasmid carrying the complementing clone. The wild type gene was sequenced and found to encode a 1268 amino acid protein of with a molecular weight of 142,655 Daltons. Computer assisted searches for similar DNA sequences revealed no significant homology matches. However, searches for protein sequence homology revealed a protein (the DIS3 gene product of S. pombe) with a similar sequence over a 534 amino acid stretch to the predicted INS1 gene product. A later search revealed a near identical sequence for a gene (SRK1) also isolated from S. cerevisiae. ^

Estrogen action during early mouse mammary gland development

Estrogens have been implicated in the normal and neoplastic development of the mammary gland. Although estradiol is essential for early mammary differentiation, its role in postnatal ductal morphogenesis is poorly defined. We have found that neonatal estradiol exposure promotes precocious ductal outgrowth and terminal end bud formation in 21 day-old female mice. In contrast to this precocious phenotype, day 21 estradiol-treated epithelium, transplanted into control host fatpads, grows more slowly than control epithelium. Western and immunohistochemical (IHC) analyses indicate that neonatally-estrogenized glands have significantly less total ER than controls at days 7 and 21, and significantly more stromal ER at day 35. Estrogen receptor α (ER) is present in the gland when treatment is initiated at day 1. We propose that the premature activation of ER by neonatal estradiol exposure, during this critical perinatal period, is a key factor in the alteration of mammary growth and ER expression. ^ To address the role of ER function in mammary morphogenesis, we have developed an in vitro system to study the effect of estradiol exposure in vivo. Keratin and ER-positive mammary epithelial cell lines from 7, 21 and 35 day-old oil or estradiol treated mice have been established. Cell lines derived from estradiol-treated mice grow significantly slower than cells from control glands. Although the level of ER expressed by each cell line is correlated to its rate of growth, epithelial growth in vitro is estradiol-independent and antiestrogen-insensitive. Estradiol-induced transcription from an ERE-reporter in transiently-transfected cell lines confirms the functionality of the ER detected by western and IHC. However, there are no differences in estradiol-stimulated transcription between cell lines. ^ In conclusion, neonatal estradiol treatment alters the pattern of ER expression in mammary epithelial and stromal cells in vivo, and the growth of mammary epithelial cells in vivo and in vitro. When grown outside of the estrogenized host, exposed epithelium grows more slowly than the control. Therefore, an extra-epithelial factor is necessary for enhanced epithelial growth. Our model, which couples an in vivo-in vitro approach, can be used in the future to identify factors involved in the period of early mammary outgrowth and carcinogen susceptibility. ^

Analysis of BMP signalling through the receptor type IA in embryogenesis using conditional gene inactivation in mice

Although bone morphogenetic proteins (BMPs) were initially identified for their potent bone-inducing activity, their precise roles in processes of endochondral and intramembranous bone formation are far from being clear. Tissue-specific loss-of-function experiments using the BMP receptor type IA (BMPR-IA) are particularly attractive since this receptor is thought to be essential for signaling by the closely related BMPs -2, 4, and 7. To ablate signaling through this receptor during chondrogenesis, we have generated transgenic mice expressing Cre recombinase under the control of the collagen type II (Col2a1) gene regulatory sequences. Mice lacking BMPR-IA function in chondrocytes display a number of skeletal abnormalities, including defects in bones of the chondrocranium, abnormal dorsal vertebral processes, scapulae with severe hypoplasia of dorsal elements, and shortening of the long bones. Alterations in the growth plate of long bones in mutants suggest that BMPR-IA is not required for early steps of the chondrocyte specification, but is rather important in regulation of terminal differentiation. Molecular analysis revealed noticeable downregulation of the Ihh/Ptch signalling pathway, decreased chondrocyte proliferation rate and deregulation of hypertrophy. ^ In order to elucidate the role of BMP signalling in development of the limb and intramembranous ossification, we have used mice expressing Cre recombinase under control of the Prx1 (MHox) regulatory elements (M. Logan, pers comm.). Cre activity was found in those mice in the developing limb bud mesenchyme, as well as in a subset of cranial neural crest cells. Prx1-Cre-induced conditional mutants display prominent defects in distal limb outgrowth, as well as ossification defects in a number of neural crest-derived calvarial bones. Intriguingly, mutant limbs displayed alterations in patterning along all three axes. Molecular analysis revealed ectopic anterior Shh/Ptch signalling pathway activation and expression of some Hox genes. Observed loss of Msx1 and Msx2 expression in the progress zone correlates with downregulation of Cyclin D1 and decreased distal outgrowth. Abnormal ventral localization of Lmx1b-expressing cells along with observed later morphological abnormalities suggest a novel role for BMP signalling in establishment or maintaining of the dorso-ventral polarity in the limb mesoderm. ^

The role of Lmx1b in the control of dorsoventral compartmentalization in the distal mesenchyme of the developing mouse limb

Lmx1b encodes a LIM-homeodomain transcription factor required for dorso-ventral (D-V) patterning in the mesenchyme of the vertebrate limb. In the absence of Lmx1b function, limbs exhibit a bi-ventral pattern indicating that Lmx1b is required for cells to adopt a dorsal cell fate. However, how Lmx1b specifies dorsal cell fates in the mesenchyme of the distal limb is unknown. Lmx1b is initially expressed throughout the dorsal and ventral limb bud mesenchyme, then becomes dorsally restricted around E10.5. At this stage, there is a sharp boundary between Lmx1b expressing and Lmx1b non-expressing cells. How the dorso-ventral Lmx1b expression boundary is formed and maintained is currently unknown. One mechanism that may contribute to establishing and/or maintaining the Lmx1b expression boundary is if the dorsal mesenchyme is a lineage-based compartment, where different groups of non-mingling cells are separated. Compartment formation has been proposed to rely on compartment-specific selector gene activity which functions to restrict cells to a compartment and specifies the identity of cells within that compartment. Based on the evidence that the dorsal limb identity relies on the expression of Lmx1b in the dorsal half of the limb mesenchyme, we hypothesized that Lmx1b might function as a dorsal limb bud mesenchyme selector gene to set up a dorsal compartment. To test this hypothesis, we developed an inducible CreERT2/ loxP based fate mapping approach that permanently marks Lmx1b wild-type and mutant cells and examined the distribution of their descendents in the developing limb. Our data is the first to show that dorso-ventral lineage compartments exist in the limb bud mesenchyme. Furthermore, Lmx1b is required for maintenance of the dorso-ventral compartment lineage boundary. The behavior of Lmx1b mutant cells that cross into the ventral mesenchyme, as well as previous chimera analysis in which mutant cells spread evenly in the ventral limb and form patches in the dorsal side, suggest that cell affinity differences prevent intermingling of dorsal and ventral cells. ^

Coordination of murine limb muscle, skeleton and connective tissue development by Lmx1b

The underlying genetic defects of a congenital disease Nail-Patella Syndrome are loss-of-function mutations in the LMX1B gene. Lmx1b encodes a LIM-homeodomain transcription factor that is expressed specifically in the dorsal limb bud mesenchyme. Gain- and loss-of-function experiments suggest that Lmx1b is both necessary and sufficient to specify dorsal limb patterning. However, how Lmx1b coordinates patterning of the dorsal tissues in the limb, including muscle, skeleton and connective tissues, remains unknown. One possibility is that each tissue specifies its own pattern cell-autonomously, i.e., Lmx1b is expressed in tissues in which it functions and different tissues do not communicate with each other. Another possibility is that tissues that express Lmx1b interact with adjacent tissues and provide patterning information thereby directing the development of tissues non-cell-autonomously. Previous results showed that Lmx1b is expressed in limb connective tissue and skeleton, but is not expressed in muscle tissue. Moreover, muscles and muscle connective tissue are closely associated during development. Therefore, we hypothesize that Lmx1b controls limb muscle dorsal-ventral (DV) patterning through muscle connective tissue, but regulates skeleton and tendon/ligament development cell-autonomously. ^ To test this hypothesis, we first examined when and where the limb dorsal-ventral asymmetry is established during development. Subsequently, conditional knockout and overexpression experiments were performed to delete or activate Lmx1b in different tissues within the limb. Our results show that deletion of Lmx1b from whole limb mesenchyme results in all dorsal tissues, including muscle, tendon/ligament and skeleton, transforming into ventral structures. Skeleton-specific knockout of Lmx1b led to the dorsal duplication of distal sesamoid and metacarpal bones, but did not affect the pattern formation of other tissues, suggesting that Lmx1b controls skeleton development cell-autonomously. In addition, this skeleton-specific pattern alteration only occurs in distal limb tissues, not proximal limb tissues, indicating different regulatory mechanisms operate along the limb proximal-distal axis. Moreover, skeleton-specific ectopic expression of Lmx1b reveals a complementary skeletal-specific dorsalized phenotype. This result supports a cell-autonomous role for Lmx1b in dorsal-ventral skeletal patterning. This study enriched our understanding of limb development, and the insights from this research may also be applicable for the development of other organs. ^

Outcomes associated with chemotherapy in elderly patients with early stage operable breast cancer

Background. Various clinical trials have proved the efficacy of adjuvant chemotherapy in women with breast cancer. Chemotherapy efficacy and guidelines for its use differ by stage of tumor and age of the patient with no clear recommendations for patients aged 70 and above. Objective. To examine the clinical and economic outcomes associated with chemotherapy use in and to examine the disparities in treatment and survival in elderly patients with early stage operable breast cancer by age and axillary node status. Methods. We studied a cohort of 23,110 node positive and 31,572 node negative women aged 65 and over diagnosed with incident American Joint Committee on Cancer (AJCC) stage I, II or IIIa breast cancer between January 1, 1991 and December 31, 2002 using SEER-Medicare data. Total patient costs were estimated using the phase of care approach and adjusted cost estimates were obtained from regression analysis using a 3% discount rate. Cox proportional hazard ratio of mortality was used to determine the effectiveness of chemotherapy. Propensity score approach was also used to minimize the bias associated with receipt of chemotherapy. To assess disparity in treatment, multivariate logistic regression analyses were performed to assess the relative odds of receiving surgery, chemotherapy and radiation after BCS for African Americans compared to Whites. Results. Regression adjusted cost estimates for all node positive patients receiving chemotherapy was approximately $2,300 and was significantly higher (p<0.05) than for patients not receiving chemotherapy. Mortality was significantly lower in node positive and node negative women aged 65-74 years receiving chemotherapy. There was a significant difference between African American and White women in receiving BCS and radiation after BCS; however this difference was explained by patient demographics, tumor characteristics and socioeconomic status (SES). African American node positive women were 21% less likely to receive chemotherapy than White women (OR, 0.79; CI, 0.68-0.92) in multivariate analysis. Conclusion. Chemotherapy is associated with increased survival in patients aged 65-74 and total costs attributable to chemotherapy differ by phase and age of the patient. Underutilization of systemic adjuvant chemotherapy in African American women requires attention and may serve as potential areas for appropriate intervention.^

Anesthetic choices and breast cancer recurrence: A retrospective cohort study of risk factors

Background: The impact of anesthetic techniques for breast cancer surgery traditionally has been centered on the incidence of acute pain syndromes and complications immediately after surgery. Evaluating anesthesia management beyond short-term effects is an emerging science. Several animal studies have concluded that regional anesthesia independently reduces cancer recurrence and metastasis. A small number of retrospective clinical studies indicate that reductions in cancer recurrence are attributable to anesthesia technique; however, individual risk factors need to be taken into consideration. ^ Purpose: The aims were to: 1) investigate differences in patient, disease and treatment factors between women who received surgical treatment for breast cancer with paravertebral regional and general anesthesia compared to women who received general anesthesia alone; 2) explore patient, disease and treatment factors associated with recurrence of breast cancer; and 3) test the association between type of anesthesia and breast cancer recurrence and survival over 22–46 months following surgery. ^ Methods: This retrospective cohort study included 358 patients with stage 0-III disease who received a partial or total mastectomy without axillary node dissection between October 2006 and October 2008 at a large academic cancer center. Follow-up ended in August 2010 with a median follow-up time of 28.8 months. ^ Results: The patient demographics were equally represented across anesthesia groups. Mean BMI (kg/m2) was greater for the patients who received general anesthesia (GA) alone (29±6.8) compared to those that received paravertebral regional block (PVB) with GA (28±5.1), p=0.001. The PVB with GA group had more advanced stages of disease (p=0.01) and longer surgeries (p=0.01) than the GA only group. Breast cancer recurrence was detected in only 1.7% of the study population. The mean age was 51±18 in those who had a recurrence compared to 58±11 in the non-recurrent group (p=0.06). Overall, no association between anesthesia type and recurrence was found (p=0.53), with an unadjusted estimated hazard ratio of 1.84 (95% CI 0.34–10.08). ^ Conclusions: In contrast to previous retrospective studies in cancer patients receiving surgical and anesthesia treatment, this study was unable to detect a difference in relating type of anesthesia with decreased breast cancer recurrence. Nonetheless, a significant association between BMI and type of anesthesia was observed and should be taken into account in future studies. Because the overall rate of recurrence was very small in this population, a larger study would be needed to detect any differences in rates of recurrence attributable to type of anesthesia. ^

Survival analysis of circulating tumor cells in triple-negative breast cancer

Background. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008. [1] Triple-negative breast cancer (TNBC) is an aggressive phenotype comprising 10–20% of all breast cancers (BCs). [2-4] TNBCs show absence of estrogen, progesterone and HER2/neu receptors on the tumor cells. Because of the absence of these receptors, TNBCs are not candidates for targeted therapies. Circulating tumor cells (CTCs) are observed in blood of breast cancer patients even at early stages (Stage I & II) of the disease. Immunological and molecular analysis can be used to detect the presence of tumor cells in the blood (Circulating tumor cells; CTCs) of many breast cancer patients. These cells may explain relapses in early stage breast cancer patients even after adequate local control. CTC detection may be useful in identifying patients at risk for disease progression, and therapies targeting CTCs may improve outcome in patients harboring them. Methods . In this study we evaluated 80 patients with TNBC who are enrolled in a larger prospective study conducted at M D Anderson Cancer Center in order to determine whether the presence of circulating tumor cells is a significant prognostic factor in relapse free and overall survival . Patients with metastatic disease at the time of presentation were excluded from the study. CTCs were assessed using CellSearch System™ (Veridex, Raritan, NJ). CTCs were defined as nucleated cells lacking the presence of CD45 but expressing cytokeratins 8, 18 or 19. The distribution of patient and tumor characteristics was analyzed using chi square test and Fisher's exact test. Log rank test and Cox regression analysis was applied to establish the association of circulating tumor cells with relapse free and overall survival. Results. The median age of the study participants was 53years. The median duration of follow-up was 40 months. Eighty-eight percent (88%) of patients were newly diagnosed (without a previous history of breast cancer), and (60%) of patients were chemo naïve (had not received chemotherapy at the time of their blood draw for CTC analysis). Tumor characteristics such as stage (P=0.40), tumor size (P=69), sentinel nodal involvement (P=0.87), axillary lymph node involvement (P=0.13), adjuvant therapy (P=0.83), and high histological grade of tumor (P=0.26) did not predict the presence of CTCs. However, CTCs predicted worse relapse free survival (1 or more CTCs log rank P value = 0.04, at 2 or more CTCs P = 0.02 and at 3 or more CTCs P < 0.0001) and overall survival (at 1 or more CTCs log rank P value = 0.08, at 2 or more CTCs P = 0.01 and at 3 or more CTCs P = 0.0001. Conclusions. The number of circulating tumor cells predicted worse relapse free survival and overall survival in TNBC patients.^

William Osler: Original Papers 1881-1897

Part 1: 1881-1888 On Some Points in the Etiology and Pathology of Ulcerative Endocarditis, 1881 On Certain Parasites in the Blood of the Frog, 1883 The Third Corpuscle of the Blood, 1883 On the Use of Arsenic in Certain Forms of Anaemia, 1886 Antifebrin, 1887 Case of Arterio-Venous Aneurism of the Axillary Artery and Vein of Fourteen Year's Duration, 1887 Typhilitis and Appendicitis, 1888 Part 2: 1889-1892 Annual Address - License to Practice 1889 Case of Syphiloma of the Cord of the Cauda Equina-Death From Diffuse Central Myelitis, 1889 On a Case of Simple Idiopathic Muscular Atrophy, Involving the Face and the Scapulo-Humeral Muscles, 1889 Note on Intra-Thoracic Growths Developing from the Thyroid Gland, 1889 On the Value of Laveran's Organisms in the Diagnosis of Malaria, 1889 On the Form of Convulsive Tic Associated with Corprolalia, Etc., 1890 A Case of Sensory Aphasia Word-blindness with Hemianopsia, 1891 Rudolf Virchow: The Man and the Student, 1891 The Healing of Tuberculosis, 1892 The Cold-Bath Treatment of Typhoid Fever, 1892 Part 3: 1893 Remarks on the Varieties of Chronic Chorea, and a Report Upon Two Families of the Hereditary Form, With One Autopsy, 1893 Note on Arsenical Neuritis Following the use of Fowler's Solution, 1893 Note on a Remarkable House Epidemic of Typhoid Fever, 1893 Cases of Sub-Phrenic Abscess, 1893 On Sporadic Cretinism in America, 1893 Notes on Tuberculosis in Children, 1893 Part 4: 1849-1895 Parotitis in Pneumonia, Case of Pericarditis Treated by Incision and Drainage, 1894 The Army Surgeon, 1894 Introductory Remarks to Course of Clinical Demonstrations on Typhoid Fever, 1894 Cancer of the Stomach with Very Rapid Course, 1895 Case of Sporadic Cretinism (Infantile Myxcedema) Treated Successfully with Thyroid Extract, 1895 Visible Contractile Tumour of the Pylorus Following Ulcer of the Stomach, 1895 On the Association of Enormous Heart Hypertrophy, Chronic Proliferative Peritonitis, and Recurring Ascites, with Adherent Pericardium, 1895 Teaching and Thinking the Two Functions of a Medical School, 1895 The Practical Value of Laveran's Discoveries, 1895 Part 5 1896 Addison's Disease, 1896 On Six Cases of Addison's Disease, 1896 Hemiplegia in Typhoid Fever Thomas Dover (of Dover's Powder) Physician and Buccaneer, 1896 John Keats The Apothecary Poet, 1896 On The Classification of the Tics or Habit Movements, 1896 The Cerebral Complication of Raynaud's Disease, 1896 Part 6: 1897 On Certain Features in the Prognosis of Pneumonia, 1897 Clinical Lecture on Mitral Stenosis - Sudden Death - Ball Thrombus in the Left Auricle, 1897 The Diagnosis of Malarial Fever, 1897 The Functions of a State Faculty (President's Address), 1897 A Clinical Lecture on The Ball-Valve Gall-Stone in the Common Duct, 1897 Pneumonia (Review of Cases studied), 1897 Internal Medicine as a Vocation, 1897 Back Notes

Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.

The yeast STM1 protein binds G*G multiplex nucleic acids in vitro and associates with ribosomes in vivo

The formation of triple helical, or triplex DNA has been suggested to occur in several cellular processes such as transcription, replication, and recombination. Our laboratory previously found proteins in HeLa nuclear extracts and in S. cerevisiae whole cell extracts that avidly bound a Purine-motif (Pu) triplex probe in gel shift assays, or EMSA. In order to identify a triplex DNA-binding protein, we used conventional and affinity chromatography to purify the major Pu triplex-binding protein in yeast. Peptide microsequencing and data base searches identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a Pu triplex-binding protein was obtained by EMSA using both recombinant Stm1p and whole cell extracts from stm1Δ yeast. Stm1p had previously been identified as G4p2, a G-quartet DNA- and RNA-binding protein. To study the cellular role and identify the nucleic acid ligand of Stm1p in vivo, we introduced an HA epitope at either the N- or C-terminus of Stm1p and performed immunoprecipitations with the HA.11 mAb. Using peptide microsequencing and Northern analysis, we positively identified a subset of both large and small subunit ribosomal proteins and all four rRNAs as associating with Stm1p. DNase I treatment did not affect the association of Stm1p with ribosomal components, but RNase A treatment abolished the association with all ribosomal proteins and RNA, suggesting this association is RNA-dependent. Sucrose gradient fractionation followed by Western and EMSA analysis confirmed that Stm1p associates with intact 80S monosomes, but not polysomes. The presence of additional, unidentified RNA in the Stm1p-immunoprecipitate, and the absence of tRNAs and elongation factors suggests that Stm1p binds RNA and could be involved in the regulation of translation. Immunofluorescence microscopy data showed Stm1p to be located throughout the cytoplasm, with a specific movement to the bud during the G2 phase of the cell cycle. A dramatically flocculent, large cell phenotype is observed when Stm1p has a C-terminal HA tag in a protease-deficient strain background. When STM1 is deleted in this background, the same phenotype is not observed and the deletion yeast grow very slowly compared to the wild-type. These data suggest that STM1 is not essential, but plays a role in cell growth by interacting with an RNP complex that may contain G*G multiplex RNA. ^