Assessment of Tumor Heterogeneity,as Evidenced by Gene Expression Profiles,Pathway Activation,and Gene Copy Number,in Patients with Multifocal Invasive Lobular Breast Tumors

BackgroundInvasive lobular carcinoma (ILC) comprises approximately ~10–20% of breast cancers. In general, multifocal/multicentric (MF/MC) breast cancer has been associated with an increased rate of regional lymph node metastases. Tumor heterogeneity between foci represents a largely unstudied source of genomic variation in those rare patients with MF/MC ILC.MethodsWe characterized gene expression and copy number in 2 or more foci from 11 patients with MF/MC ILC (all ER+, HER2-) and adjacent normal tissue. RNA and DNA were extracted from 3x1.5mm cores from all foci. Gene expression (730 genes) and copy number (80 genes) were measured using Nanostring PanCancer and Cancer CNV panels. Linear mixed models were employed to compare expression in tumor versus normal samples from the same patient, and to assess heterogeneity (variability) in expression among multiple ILC within an individual.Results35 and 34 genes were upregulated (FC>2) and down-regulated (FC<0.5) respectively in ILC tumor relative to adjacent normal tissue, q<0.05. 9/34 down-regulated genes (FIGF, RELN, PROM1, SFRP1, MMP7, NTRK2, LAMB3, SPRY2, KIT) had changes larger than CDH1, a hallmark of ILC. Copy number changes in these patients were relatively few but consistent across foci within each patient. Amplification of three genes (CCND1, FADD, ORAOV1) at 11q13.3 was present in 2/11 patients in both foci. We observed significant evidence of within-patient between-foci variability (heterogeneity) in gene expression for 466 genes (p<0.05 with FDR 8%), including CDH1, FIGF, RELN, SFRP1, MMP7, NTRK2, LAMB3, SPRY2 and KIT.ConclusionsThere was substantial variation in gene expression between ILC foci within patients, including known markers of ILC, suggesting an additional level of complexity that should be addressed.     

Immunocytochemical localization of somatostatin and vasotocin in the brain of the Pacific hagfish,Eptatretus stouti

Summary The brain of the Pacific hagfish, Eptatretus stouti, was studied immunocytochemically using antisera against somatostatin (SRIH), arginine vasopressin (AVP), and adrenocorticotropic hormone (ACTH). SRIH-immunoreactive perikarya were distributed bilaterally in the postoptic nucleus and in the hypothalamic nucleus. Although several short, stained fibers were observed in the vicinity of the perikarya, SRIH-immunoreactivity was not found in the neurohypophysis, nor in other parts of the brain. On the other hand, presumed arginine vasotocin (AVT) perikarya were distributed in an arc-shaped region extending from the posterior part of the preoptic nucleus to the anterior-most end of the hypothalamic nucleus and projected their fibers to the neurohypophysis. Most presumptive AVT perikarya were located close to the paired prehypophysial arteries near the anterior end of the postoptic nucleus. In the neurohypophysis, abundant presumptive AVT-fibers terminated in the posterior dorsal wall, although some fibers terminated in the anterior dorsal wall and only a few fiber endings were found in the ventral wall. No ACTH-positive cells were detected in the hagfish brain or in the pituitary gland.Supported from a grant from the National Science Foundation PCM 8141393     

Purification and general properties of argininosuccinate lyase from jack bean, Canavalia ensiformis (L.) DC

1. Argininosuccinate lyase (EC 4.3.2.1) from jack bean [Canavalia ensiformis (L.) DC] seeds was purified 532-fold from an acetone-butanol-dried powder. 2. The enzyme functions reversibly and exhibits maximum stability at 16 degrees . 3. At 16 degrees it has a half-life (t((1/2))) of 263min. 4. The enzyme is both cold-labile (t((1/2)) 131min. at 0 degrees ) and heat-inactivated (t((1/2)) 74min. at 38 degrees ); inactivation appears to be irreversible. 5. Treatment of the acetone-butanol-extracted powder with sodium dodecyl sulphate increased the sensitivity of the enzyme to temperature (t((1/2)) 70min. at 0 degrees ; t((1/2)) 23min. at 38 degrees ). 6. Addition, to the purified enzyme, of a fraction containing lipid from the seed increased the half-life to about 510min. at either 0 degrees or 38 degrees . 7. Arginine or homoarginine, and to a smaller extent some other amino acids or fumarate, protected the enzyme from cold-inactivation. 8. Reactivation attempts with both the cold- and heat-inactivated enzyme failed. 9. The K(m) value for argininosuccinate at pH7.5 is 1.3x10(-4). 10. The enzyme was inactivated completely within 15min. at 16 degrees by 0.5mm-p-hydroxymercuribenzoate, and subsequent exposure to 5mm-cysteine had no restorative effect.     

Physical and chemical studies of a low-molecular-weight form of urease

1. A new form of enzymically active jack-bean [Canavalia ensiformis (L.) DC] urease corresponding to an S_20,w value of 11·8s and a molecular weight of 260000 was investigated. 2. Conversion of 18s urease (EC 3.5.1.5) into the 12s form depends on both low protein concentration and pH. Above pH5·3 urease exists in the 18s form and below pH4·8 in the 12s form; between these two pH values a 12s–18s rapid-equilibrium process is observed. 3. Comparison of the properties of 18s and 12s urease indicated no major differences. 4. A survey of other good urease sources revealed that the 12s form can also be obtained from soya bean (Glycine soja Sieb. and Zucc. cultivar Biloxi) and the bacterium Bacillus pasteurii (Miguel) Migula, but not from watermelon (Citrullus vulgaris Schrad. cultivar Congo). 5. The 12s forms from jack bean and Bacillus pasteurii did not hybridize.     

Hypodontia: Prevalence amongst Jewish populations of different origin

The teeth of 10,371 male and 11,013 female Israel Jews were examined. Prevalence of all hypodontia was 4.60% with no significant difference between the sexes; 2.11% lacked upper lateral incisors, the females having a significantly higher prevalence than males. Second premolars were missing in 1.87% of the population, with no significant differences between the sexes. Missing lower incisors was diagnosed in 0.68% of the children, with a higher prevalence in males. Prevalence of missing lower incisors was similar in the Ashkenazi and in the non-Ashkenazi. The teeth most frequently missing in descending order were the upper lateral incisors and the lower second premolars.     

Plant growth with new fluorescent lamps

Summary Bean and marigold plants were grown to maturity under several kinds of fluorescent lamps to evaluate the effects of spectral differences on development and reproduction. Six kinds of lamps were tested including five lamps that were used in closely related experiments on tomato seedling growth (Thomas and Dunn, 1967). Evaluation was by fresh- and dry-weight yields of immature and mature pods, and of vegetative tops of plants for bean; and by flowering and fresh-and dry-weight yields for marigold.Bean plants grown under two experimental lamps, Com I and IR III produced significantly higher fresh- and dry-weight yields of both mature and total pods than under Warm-white lamps. This effect could be attributed largely to the considerable energy emitted by the experimental lamps in the red and far-red, as compared to a larger emission in the green and blue for the Warm-white lamps. The differences in the yields for immature pods and vegetative portions of the mature tops were not significant.In a comparison of the effects of three experimental lamps with those of three commercial lamps on growth response of bean plants, the yields were in general higher for the experimental lamps, except for immature pods. The yields of vegetative tops were significantly greater for the 78/22 lamp over the yields for all other lamps. The larger proportion of red and far-red light emitted by the experimental lamps is again the probable cause of the higher yields with these lamps.Two sets of experiments on growth and flowering of marigold under various experimental and commercial lamps were largely inconclusive although there was some indication of beneficial effects by the experimental lamps.In general, the results with bean agree with those for tomato (Thomas and Dunn, 1967), in that best growth was obtained with a lamp high in red light emission, a moderate amount in the far-red, and very little in the blue part of the spectrum.This research was submitted by the senior author in partial fulfillment of the requirements for the M.S. degree in Botany at the University of New Hampshire.Published with the approval of the director of the New Hampshire Agricultural Experiment Station as Scientific Contribution No. 398. This study was part of the Northeast Regional Project, NE-35, Analysis of Northeastern Climatic Variables and Their Relationships to Plant Response.     

Proline at position 36: a new transthyretin mutation associated with familial amyloidotic polyneuropathy.

Familial amyloidotic polyneuropathy (FAP) is associated with the deposition of an abnormal transthyretin (TTR) molecule. We have studied DNA from a family of Greek descent with FAP. The proband's TTR gene was asymmetrically amplified by using PCR and then was sequenced directly, to reveal a cytosine-for-guanine substitution in codon 36. This substitution removes a recognition site for endonuclease Fnu4HI. Allele-specific PCR was employed for diagnosis of the mutation. The predicted amino acid change of alanine to proline at position 36 was confirmed by protein sequencing of the proband's plasma TTR.     

Localization of the genes for histatins to human chromosome 4q13 and tissue distribution of the mRNAs.

A cDNA coding for histatin 1 was isolated from a human submandibular-gland library and sequenced. This cDNA was used to probe RNAs isolated from a variety of tissues to investigate tissue-specific regulation and to determine whether histatins might play a role other than in the oral cavity. The same probe was also used for Southern blot analysis of human genomic DNA restricted with various enzymes, and it showed that the genes coding for histatins are on the same chromosome. In situ hybridization of the cDNA probe to metaphase chromosome spreads was performed to determine chromosomal location of the genes for histatins. A genomic fragment isolated using the cDNA probe was also hybridized to chromosome spreads, and the same chromosome was identified. The genes for histatins are located on chromosome 4, band q13. We have shown that three histatin mRNAs are expressed in human parotid and submandibular glands but in none of the other tissues studied. These results suggest that histatins are specific to salivary secretions.     

Saturation Binding of Nicotine to Synthetic Neuromelanin Demonstrated by Fluorescence Spectroscopy

Neuromelanin (NM) has long been considered as an aging pigment, perhaps an unavoidable and undesirable byproduct of dopaminergic neural transmission. However, NM is carefully packaged into double membrane-bound structures within cells of the substantia nigra and other neural tissues, suggesting a beneficial function to maintaining these stores. It is well established that NM is able to concentrate toxic xenobiotics within pigmented cells due to its unique chemical environment. In doing so, such agents may confer susceptibility to Parkinson’s disease (PD) as illustrated by model PD-inducing neurotoxins such as methyl-phenyl-pyridinium ion. It is possible that high-affinity binding interactions toward NM may contribute to the adverse effects of PD-inducing toxins, as well as neuroprotective agents. Here we aim to develop a generalized assay capable of elucidating the binding constants of chemical agents to synthetic and natural neuromelanins. Toward this end, a model neuromelanin synthesized from dopamine and cysteine was prepared according to published procedure. Using a UV/Visible spectroscopic assay, we show that dopamine, 6-hydroxy dopamine, and nicotine bind to the synthetic neuromelanin, while caffeine did not. More importantly, nicotine was further found to induce a fluorescence signal in the presence of NM which was used to establish a binding constant estimated at 0.65 mM. Dopamine appears to enhance this signal, also in a saturable manner, with an estimated Kd of 0.05 mM in our isolated chemical system. In summary, the micro-scale fluorescence assay described herein will allow us to overcome many of the problems inherent in the study of chemical interaction with NM through traditional spectroscopic means. Using a single standardized signal, it should now be possible to rank a number of PD-related toxins based on NM-binding affinity and shed further light on this important problem.