Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Dual role of osteoblastic proenkephalin derived peptides in skeletal tissues

Proenkephalin encodes a group of small peptides with opiate-like activity, the endogenous opioids, known to function as neurohormones, neuromodulators, and neurotransmitters. Recently, we have demonstrated that in addition to its abundance in fetal brain tissue, proenkephalin is highly expressed in nondifferentiated mesodermal cells of developing fetuses. We identified the skeletal tissues, bone, and cartilage as major sites of proenkephalin expression. To examine the possibility that proenkephalin is involved in bone development we have studied the expression of this gene in bone-derived cells, its modulation by bone active hormones, and the effects of enkephalin-derived peptides on osteoblastic phenotype. Our studies revealed that osteoblastic cells synthesize high levels of proenkephalin mRNA which are translated, and the derived peptides are secreted. Reciprocal interrelationships between osteoblast maturation and proenkephalin expression were established. These results together with our observations demonstrating inhibitory effects of proenkephalin-derived peptides on osteoblastic alkaline phosphatase activity, strongly support the notion that proenkephalin is involved in bone development. A different direction of research by other investigators has established the capability of the opioid system in the periphery to participate in the control of pain. On the basis of these two lines of observation, we would like to present the following hypothesis: The potential of embryonic skeletal tissue to synthesize proenkephalin-derived peptides is retained in the adult in small defined undifferentiated cell populations. This potential is realized in certain situations requiring rapid growth, such as remodeling or fracture repair. We suggest that in these processes, similarly to the situation in the embryo, the undifferentiated dividing cells produce the endogenous opioids. In the adult these peptides may have a dual function, namely participating in the control of tissue regeneration and in the control of pain. © 1994 Wiley-Liss, Inc.     

Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: A spectroscopic study

Summary Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.     

Rhythmic signalling and entrainment inVespa orientalis larvae: Characterization of the underlying interactions

Rhythmic “circa-second” contrations of larvae of the hornetVespa orientalis, believed to serve as hunger signals, were studied. A considerable degree of coordination among individual larvae, both in frequency and phase of these contractions, has been observed. The oscillations of singly isolated larvae are of short duration, non-constant, with increasing intervals in between and there is a substantial variability in the patterns shown by different larvae. In contrast, the association of two or more larvae leads to enhancement of their periodic behaviour and to (partial) entrainment. Communication among larvae may perhaps be mediated by the sound pulses (“scratching” noises) which are generated by these contractions. We have subjected individual and grouped larvae to external sound pulses and were able to demonstrate: (a) enhancement of rhythmic activity; (b) phase resetting; (c) entrainment to an external oscillator within a range of frequencies; (d) the existence of a subharmonic mode of entrainment. We propose a simple phenomenologic model to account for these larvae responses. Our model assumes the existence of an “energy” variable which declines with time but is upgraded, in a phase-dependent way, by external stimuli. Based in part on work performed by V. Barenholz-Paniry in partial fulfillment of the requirements for the M.Sc. degree from the Sackler Faculty of Medicine, Tel Aviv University, 1986.     

Frontiers in mycoplasma pathogenicity

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss₁ and Yop₁₂) andStreptococcus salivarius subsp.thermophilus (Ss₂ and Yop₉) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop₁₂+Ss₂) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.     

Characterisation of P. falciparum antigenic determinants isolated from a genomic expression library by differential antibody screening.

A genomic expression library of P.falciparum has been differentially screened with a number of immune sera. The response of 9 clones to the various sera is presented, together with the DNA sequence encoding the epitopes. All but one clone are extremely A+T rich and unlike the other P.falciparum epitopes described, are not composed of amino acid repeats. One clone, which responds specifically with a protective serum, has been analysed in detail. The epitope is carried on a 160kd antigen which is transcribed from a single gene to give a protein expressed in all of the erythrocytic forms. DNA sequence of this clone reveals it to have more than one open reading frame, only one of which is transcribed in the blood stages. The possible significance of the other open readings frames is discussed.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

The mechanism of photodynamic inactivation ofStaphylococcus aureus by deuteroporphyrin

The mechanism ofStaphylococcus aureus inactivation by deuteroporphyrin (DT) and light was studied with singlet oxygen quenchers or hydroxyl radical scavengers. The light-activated DT (10 /ml) reduced the viability of the culture to less than 1%, whereas methionine, tryptophan, and 1,4-diazabicyclo-2,2,2-octane (DBCO) used as singlet oxygen quenchers provided almost 60% protection. Propylgallate, which is a hydroxyl free radical scavenger, also provided 60% protection. The presence of a singlet oxygen quencher and propylgallate provided almost complete protection from inactivation (96%). Photoinactivation in the absence of culture media (in saline) increased the killing rate and decreased the ability of the singlet oxygen quenchers to protect. In the same conditions damage from hydroxl free radicals was well protected by propyl gallate. The present results indicate thatS. aureus photoinactivation by DT and light is mediated by both singlet oxygen and hydroxyl free radicals.     

Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gsα gene

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gs, the subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2–13 of the Gs gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albright's hereditary osteodystrophy, resistance to multiple hormones, and deficient Gs activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gs. This report adds two new Gs mutations to the known ten mutations recently described.