Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Urinary cortisol analysis for monitoring adrenal activity in elephants

Cortisol was measured in dichloromethane-extracted elephant urine using an ¹²⁵I solid-phase radioimmunoassay (RIA). The cortisol RIA was validated by demonstrating 1) parallelism between dilutions of pooled urinary extracts and the standard curve, 2) significant recovery of exogenous cortisol added to elephant urine, and 3) a relationship between changes in peripheral and urinary cortisol after an adrenocorticotropin hormone (ACTH) challenge. One African (Loxodonta africana) and one Asian (Elephas maximus) elephant were given three injections of ACTH (1.25 mg) at 2 h intervals. Serum cortisol increased four- to eightfold within 30 min after the first injection and peaked (nine- to twelvefold increase) after the second injection. Serum concentrations began to decline 2–3 h after the last injection but were still approximately fourfold higher than baseline at the end of the collection period (hour 8). In the urine, cortisol concentrations were increased in the first sample postinjection (1.5–4 h) and peaked twenty- to fortyfold by ～6 h. Urinary cortisol remained elevated at 8 h, but returned to baseline the following morning. Analysis of high performance liquid chromatography fractions of extracted urine revealed that immunoactivity was associated with free cortisol (～90% of total immunoactivity) and a more polar, unidentified metabolite. A method for preserving urine was developed to allow storing unfrozen samples. One pool of urine from each of one African and two Asian elephants was divided into aliquots, placed in tubes containing absolute ethanol (10%), sodium azide (0.1%) or distilled water (control), and frozen after 0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 weeks of storage at ～25°C. In unpreserved samples, cortisol concentrations were reduced 46% by 2 weeks and 95% by 24 weeks. In contrast, ethanol- and sodium azide-preserved samples retained 100 and 95% of cortisol immunoactivity through 8 weeks and 93 and 85% of activity through 12 weeks, respectively. We infer from these data that changes in urinary cortisol excretion in the elephant reflect fluctuations in adrenal activity and may be a useful indicator of stress. Additionally, urine samples can be collected and stored unfrozen for at least 2 months before any appreciable loss in cortisol immunoactivity occurs, a finding potentially useful to field application of this technique. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

.     

Incorporation of 5-bromo-2′deoxyuridine (BUdR) in Dictyostelium discoideum DNA

The modality of incorporation of 5-bromo-2′deoxyuridine (BUdR) in the DNA of Dictyostelium discoideum was studied after one generation of growth of the amoebae in the presence of different concentrations of the drug. The analog was incorporated following the semiconservative pattern of DNA replication. BUdR incorporation in monosubstituted DNA has been measured both by CsCl isopycnic centrifugation or by base analysis chromatography; substitution of thymidine by its analog reaches a maximal value of 30% (60% in the substituted strand). Up to 20% substitution it is proportional to the drug concentration in the growth medium. In these conditions, thymidine substitution is higher in repetitive sequences of the DNA than in unique sequences; the percent of increase of thymidine substitution in repetitive fractions versus total DNA is inversely proportional to thymidine substitution in total DNA.     

T lymphocytes responding to Mls-locus antigens are Lyt-1+, 2− andI-A restricted

We have investigated primary and secondary responses of mouse splenic T cells to strong mixed lymphocyte stimulating antigens controlled by theMls locus using MHC-identical mixtures of cells. Our studies show that strong primaryMls-locus specific responses involve recognition of self I-A antigens, since BUdR and light suicide or F1 into parent radiation bone-marrow chimeras both demonstrate a preference of unprimed F1 T cells to respond to Mis-locus antigens associated with one parent's MHC antigens. Furthermore, conventional anti-I-A antisera and monoclonal anti-I-A antibody both inhibitMls-locus responses in an MHC-specific manner. Finally, as is typical of T cells responding to I-A antigens or to nominal antigens associated with self I-A,Mlslocus responses are mediated by Lyt-1⁺, 2^– cells. One striking finding in these studies was the very high frequency of cells capable of responding to Mls-locus antigens, the highest being 1/300 splenic T cells. This plus evidence for recruitment during primaryMls-locus responses may account for reports of a lack ofI-A restriction in secondary anti-Mls locus responses to strong Mls-locus antigens, a finding with which we concur. The possibility that these secondary responses between noncongenic strains of mice may be directed at other genetic loci is also discussed. These experiments leave open the question of the biological role of theMls-locus and of the very large number of T cells reactive to it.Abbreviations used in this paper MHC Major histocompatibility complex - MIg Mouse immunoglobulin - MLC Mixed lymphocyte culture - TCGF T-cell growth factor     

Intraspecific host discrimination and larval competition inMicroplitis croceipes,Microplitis demolitor,Cotesia kazak (HYM.: Braconidae) andHyposoter didymator HYM: Ichneumonidae), parasitoids ofHeliothis virescens (LEP.: Noctuidae)

Intraspecific host discrimination and larval competition were studied forMicroplitis croceipes (Cresson),Microplitis demolitor Wilkinson,Cotesia kazak (Telenga), andHyposoter didymator (Thunberg), solitary endoparasitoids of the tobacco budworm,Heliothis virescens (F.). In ovipositional choice tests between unparasitized and parasitized hosts, the mean number of ovipositions for unparasitized hosts was significantly higher than the mean number of ovipositions for hosts parasitized once by a conspecific female forC. kazak andH. didymator, demonstrating that females of these two species discriminate against hosts recently (within a few seconds) parasitized by a conspecific female. No significant difference in oviposition occurred between these two kinds of hosts forM. croceipes andM. demolitor. Mean percent parasitization by a second conspecific female was determined at 24, 48, and 72 h delays in time between the first and second female attack, and with no delay. Except for the 0 h time delay forC. kazak andH. didymator, percent parasitization by a second conspecific female generally decreased as the delay in time between the first and second female attack increased. When the second parasitization immediately followed the first, one parasitoid larva always eliminated the other by physical combat. With a 24 or 48 h delay between the first and second parasitization, the younger larva was the victor over the older larva forM. croceipes, M. demolitor andC. kazak in at least 50% of the cases. Elimination of older larvae by younger larva was by physical attack. However, forH. didymator, the older instar was the victor, and elimination of younger larvae by older larvae was probably through physiological processes. Further, older larvae ofH. didymator apparently killed the eggs of the second female by physiological processes.      

Paléoxylologie: Étude anatomique comparée de Scleromedulloxylon aveyronense nov. gen. nov. sp. du Permien de St-Affrique (Aveyron,France); considérations taxinomiques et stratigraphiques

The fossil woods studied in the present paper were collected by P. Béziat in the Permian Basin of St-Affrique (Southern Aveyron). They come from near Calmels. They are the first fossil woods collected and described in this basin.The secondary wood refers to the form genus Dadoxylon and more exactly to the speciesDadoxylon schrollianum (Goeppert, 1864–65) Frentzen, 1931, Frentzen, 1931. The pith is certainly related with the pith structure of Walchiapremnon valdajolenseFlorin, 1940.The association of a Dadoxylon secondary wood with an irregularly septate pith containinggroups of probably sclerotic cells can be found in Cedroxylon varollenseRenault, 1893–1896, Renault and Roche, 1894 from the Basin of Autun. It is not possible to conserve the species varollense in the genus Cedroxylon and we propose the form genus Scleromedulloxylon for the petrified structure from Autun and for the fossil woods of St-Affrique.Furthermore, a comparative anatomical study of the different Permo-Carboniferous structuresfrom the Euramerican province let us, when it is possible, distinguish structure-types related to Cordaitophyts or to Coniferophyts. In the present case, Scleromedulloxylo should rather correspond to the fossil wood of an Autunian Coniferophyt, that is to Walchia structure.     

Ring‐opening of azetidiniums by nucleophiles. Synthesis of polysubstituted linear amines

This microreview focuses on the nucleophilic ring‐opening of azetidiniums presenting various substitution patterns at C2, C3, and C4. In most cases, the nucleophilic ring‐opening occurred in a stereoselective and regioselective fashion producing functionalized linear amines. Experimental selectivities associated with Density Functional Theory (DFT) calculations have allowed a better understanding of the parameters governing the regioselectivities.