Structural and functional variability among DQ β alleles of DR2 subtypes

Homozygous lymphoblastoid cell lines representing various Dw subtypes of DR2 were examined for polymorphism at the DQ locus by molecular and cellular techniques. The subtypes studied included Dw2, Dw12, and a group heterogenous by cellular typing that we shall refer to as non-Dw2/non-Dw12. Restriction fragment length polymorphism analysis of cell lines representing these subtypes revealed DQ -specific patterns consistent with cellular typing. Two-dimensional gel electrophoresis of DQ molecules from representative cell lines revealed a structural polymorphism of DQ among the three subtypes. The DQ chain migrated to a position that was unique to each subtype and was consistent among various representative cell lines of each subtype. Nucleotide sequence analysis of cDNA clones of DQ from Dw2, Dw12, and non-Dw2/non-Dw12 lines confirmed that the variability resided at the genetic level. Variability was found in the form of numerous scattered nucleotide substitutions throughout the first domain of these alleles. The DQ gene of the non-Dw2/non-Dw12 cell line AZH was further found to be almost identical with the DQ gene of a DR1 line (Bell et al. 1985b), implicating a common evolutionary origin of these alleles. The only difference between these two sequences was due to an apparent gene conversion event at amino acid 57. T-cell cloning experiments resulted in the derivation of Epstein-Barr virus-specific, DQw1-restricted clones that proliferated against only those cell lines that exhibited the DQ gene common to AZH and the DR1 cell line. Thus, the polymorphism among DQ alleles within DR2 results in subtype-specific restriction.     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

Prognostic significance of DNA ploidy and morphometric analyses of adenocarcinoma of the uterine cervix

In an effort to improve the prognostic accuracy of the histologic criteria used for cervical adenocarcinomas, the nuclear DNA ploidy levels, means and standard deviations of nuclear areas and amounts of lumen and neoplastic tissue were quantitated. Useful thresholds in discriminating recurrent disease, as identified by logistic regression analysis, included a DNA ploidy level of 3.0 N, a percent of lumen of 34.6% and nuclear area mean and standard deviation of 53.1 sq micron and 20.1 sq micron, respectively. These parameters should provide useful guidelines in the visual assessment of histologic features that have prognostic significance.     

The effect of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus

Synopsis The effects of constant and diurnally fluctuating levels of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus, were examined under controlled laboratory conditions. Fish were exposed for either 10 or 11 weeks to constant levels of 6.7 (high) and 2.2 (low) mg l^–1, and a diurnal fluctuation, ranging from 2.5 to 6.4 mg 0₂l^–1. Growth rates, calculated for both standard length and weight, for fish exposed to low and diurnally fluctuating levels were significantly reduced (p < 0.001) as compared to those for fish exposed to the high level. Growth rates of fish exposed to the high level were over twice those of fish held under low oxygen conditions. Under fluctuating conditions, fish grew at intermediate rates. Following these exposures, all fish were subsequently held at 7.2 mg Oz l^–1 for five weeks. Growth rates increased over two and a half times for fish previously exposed to the low oxygen level and were significantly (p < 0.001) higher than for the other two groups.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

Molecular cloning of the F8 fimbrial antigen from Escherichia coli

Abstract The genetic determinant coding for the P-specific F8 fimbriae was cloned from the chromosome of the Escherichia coli wild-type strain 2980 (O18:K5:H5:F1C, F8). The F8 determinant was further subcloned into the Pst I site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned counterpart was demonstrated. The cloned F8 fimbriae and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepithelial cells. The cloned F8 determinant was well expressed in a variety of host strains.     

A nonlethal mutation in large T antigen of polyomavirus which affects viral DNA synthesis.

A mutation in polyomavirus large T antigen which affects viral DNA synthesis was discovered in strain NG59RA (RA). The effect was most visible in nonpermissive cells. Although a substantial yield in DNA synthesis is normally observed in infections of Fischer rat cells when these are maintained at 33 degrees C (D.L. Hacker, K.H. Friderici, C. Priehs, S. Kalvonjian, and M.M. Fluck, p. 173-181, in R.E. Moses and W.C. Summers, ed., DNA Replication and Mutagenesis, 1988; D.L. Hacker and M.M. Fluck, Mol. Cell. Biol., in press), a 10- to 20-fold decrease in yield was obtained in infections with RA. The yield of free viral DNA in RA transformants was also strongly diminished, whether the transformants were maintained at 37 or 33 degrees C. A large reduction in the apparent number of integration sites, as well as a small reduction in the incidence of tandem integration of the viral genome, was observed in F-111 or FR-3T3 cells transformed by the mutant strain. This appears not to be directly related to the number of integration templates. A DNA fragment was identified which rescues these phenotypes. The fragment is located between the HindIII and NsiI restriction sites (nucleotides 1656 to 1910), a region which encodes only large T antigen. Sequence analysis of this region reveals a C-to-G transition at nucleotide 1791 which causes a proline-to-alanine change in the amino acid sequence of large T antigen. No other mutations have been previously reported in this region of large T antigen.