Mutagenic Analysis of the Intracellular Portals of the Human 5-HT3A Receptor

Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg⁴³⁶ and Arg⁴⁴⁰) within the MA helix of 5-hydroxytryptamine type 3A (5-HT₃A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT₃A receptor model. We probed the majority of residues within the MA helix of the human 5-HT₃A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT₃A receptor portals, which agrees well with the homology model.     

The chromosomes of Schoutedenia lutea (homoptera,aphidoidea, greenideinae), with an account of meiosis in the male

Somatic chromosomes of both sexes and chromosome behaviour during spermatogenesis were studied in the aphid Schoutedenia lutea (van der Goot). Four long but unequal chromosomes in females were interpreted as X chromosomes (X₁X₁X₂X₂) with one member of an autosome pair attached to one X₁, and the other member to one X₂, so that the four long chromosomes were actually X₁+A, X₁, X₂+A, X₂. Males (normally XO in aphids) received X chromosomes corresponding in relative length to the two longest (X₁+A, X₂+A) in females. During spermatogenesis parallel pairing occurred in prophase 1 and the X₁ and X₂ chromosomes became associated via their autosomal segments. In anaphase I, the autosomal segment became detached from one of the X chromosomes and entered the non-viable (non-X-bearing) spermatocyte, while the viable spermatocyte received both X₁ and X₂ (either one of which still carried an autosome) and the haploid set of free autosomes. The consequences for sex determination and zygote formation of this unusual system are discussed; a stable chromosomal constitution for the zygote can be achieved only at the expense of considerable gamete wastage.     

GENETIC EVIDENCE FOR THE OCCURRENCE OF A NEW SPECIES OF NEOPHYLLAPHIS TAKAHASHI (HOMOPTERA: APHIDIDAE) IN AUSTRALIA

Abstract
Chromosomal and electrophoretic evidence is presented demonstrating the occurrence of a fourth species of Neophyllaphis Takahashi in Australia. The new species, N. lanata sp. n., occurs on Podocarpus spinulosus. Karyotypes of all four species of Neophyllaphis in Australia are provided.     

Analysis of adenovirus type 2 L1 RNA 3''-end formation in vivo and in vitro.

Downstream sequence requirements for efficient cleavage and polyadenylation at the adenovirus type 2 L1 poly(A) site were determined in vivo in 293 cells and in vitro by using RNA precursors in HeLa cell nuclear extracts. The two cleavage sites used were found to differ in sensitivity to 3'-end deletion in vivo and in vitro.     

Relation of infant feeding to adult serum cholesterol concentration and death from ischaemic heart disease.

OBJECTIVE--To examine whether method of infant feeding is associated with adult serum lipid concentrations and mortality from ischaemic heart disease. DESIGN--Follow up study of men born during 1911-30. SETTING--Hertfordshire, England. SUBJECTS--5718 men, for 5471 of whom information on infant feeding had been recorded by health visitors and 1314 of whom had died. 485 of the men born during 1920-30 and still living in Hertfordshire who had blood lipid measurements. MAIN OUTCOME MEASURES--Death from ischaemic heart disease; serum cholesterol and apolipoprotein concentrations. RESULTS--474 men had died from ischaemic heart disease. Standardised mortality ratios were 97 (95% confidence interval 81 to 115) in men who had been breast fed and had not been weaned at 1 year, 79 (69 to 90) in breast fed men who had been weaned at 1 year, and 73 (59 to 89) in men who had been breast and bottle fed. Compared with men weaned before one year men not weaned had higher mean serum concentrations of total cholesterol (6.9 (not weaned) v 6.6 (weaned) mmol/l), low density lipoprotein cholesterol (5.0 v 4.6 mmol/l) and apolipoprotein B (1.14 v 1.08 g/l). Men who had been bottle fed also had a high standardised mortality ratio for ischaemic heart disease (95; 68 to 130) and high mean serum concentrations of total cholesterol (7.0 mmol/l), low density lipoprotein cholesterol (5.1 mmol/l), and apolipoprotein B (1.14 g/l). In all feeding groups serum apolipoprotein B concentrations were lower in men with higher birth weight and weight at 1 year. CONCLUSIONS--Age of weaning and method of infant feeding may influence adult serum low density lipoprotein cholesterol concentrations and mortality from ischaemic heart disease. Adult serum apolipoprotein B concentrations are related to growth in fetal life and infancy.     

Increased postimplantation loss and malformations among the F2 progeny of male rats chronically treated with cyclophosphamide.

Cyclophosphamide, administered to the male rat, produces increased pre- and postimplantation loss in the progeny as well as an increase in the numbers of malformed and growth retarded fetuses. The purpose of this study was to determine whether the adverse effects of chronic paternal cyclophosphamide exposure are transmissible to the next generation, the F2 progeny. Adult male rats were treated by gavage daily with saline or with cyclophosphamide (3.4 or 5.1 mg/kg) for 4 or 18 weeks and mated. The male and female offspring in each treatment group (F1 generation) were randomly mated. The resulting pregnant females were killed on day 20 of gestation to evaluate progeny outcome in the F2 generation. There was a significant increase in postimplantation loss among the offspring of the group whose fathers had been treated with cyclophosphamide at a dose of 5.1 mg/kg/day. Exposure to a dose of 5.1 mg/kg/day of cyclophosphamide also resulted in an F2 generation with a significantly decreased mean fetal weight per litter and a significant increase in the number of malformed fetuses. The malformations observed among the F2 progeny included open eyes, omphalocele, generalized edema, syndactyly, gigantism, and dwarfism. Thus, exposure of the father to cyclophosphamide does result in a specific and heritable alteration in the fertility of the surviving "apparently normal" F1 progeny. Interestingly, the adverse consequences of exposure of male rats to cyclophosphamide are similar in the F2 generation to those previously reported for the F1 progeny.     

Chimpanzee fetal G gamma and A gamma globin gene nucleotide sequences provide further evidence of gene conversions in hominine evolution

The fetal globin genes G gamma and A gamma from one chromosome of a chimpanzee (Pan troglodytes) were sequenced and found to be closely similar to the corresponding genes of man and the gorilla. These genes contain identical promoter and termination signals and have exons 1 and 2 separated by the conserved short intron 1 (122 bp) and exons 2 and 3 separated by the more rapidly evolving, larger intron 2 (893 bp and 887 bp in chimpanzee G gamma and A gamma, respectively). Each intron 2 has a stretch of simple sequence DNA (TG)n serving possibly as a "hot spot" for recombination. The two chimpanzee genes encode polypeptide chains that differ only at position 136 (glycine in G gamma and alanine in A gamma) and that are identical to the corresponding human chains, which have aspartic acid at position 73 and lysine at 104 in contrast to glycine and arginine at these respective positions of the gorilla A gamma chain. Phylogenetic analysis by the parsimony method revealed four silent (synonymous) base substitutions in evolutionary descent of the chimpanzee G gamma and A gamma codons and none in the human and gorilla codons. These Homininae (Pan, Homo, Gorilla) coding sequences evolved at one-tenth the average mammalian rate for nonsynonymous and one-fourth that for synonymous substitutions. Three sequence regions that were affected by gene conversions between chimpanzee G gamma and A gamma loci were identified: one extended 3' of the hot spot with G gamma replaced by the A gamma sequence, another extended 5' of the hot spot with A gamma replaced by G gamma, and the third conversion extended from the 5' flanking to the 5' end of intron 2, with G gamma replaced here by the A gamma sequence. A conversion similar to this third one has occurred independently in the descent of the gorilla genes. The four previously identified conversions, labeled C1-C4 (Scott et al. 1984), were substantiated with the addition of the chimpanzee genes to our analysis (C1 being shared by all three hominines and C2, C3, and C4 being found only in humans). Thus, the fetal genes from all three of these hominine species have been active in gene conversions during the descent of each species.      

Regulation of calcium-activated nonselective cation channel activity by cyclic nucleotides in the rat insulinoma cell line,CRI-G1

The regulation of a calcium-activated nonselective cation (Ca-NS⁺) channel by analogues of cyclic AMP has been investigated in the rat insulinoma cell line, CRI-G1. The activity of the channel is modulated by cyclic AMP in a complex way. In the majority of patches (83%) tested concentrations of cyclic AMP of 10 μm and above cause an inhibition of channel activity which is immediately reversible on washing. In contrast, lower concentrations of cyclic AMP, between 0.1 and 1.0 μm, produce a transient activation of channel activity in most patches (63%) tested. One group of analogues, including N⁶-monobutyryl cyclic AMP and N⁶, 2′-O-dibutyryl cyclic AMP reduced the activity of the Ca-NS⁺ channel at all concentrations tested and 2′-O-Monobutyryl cyclic AMP produced inhibition in all patches tested except one, at all concentrations. A second group produced dual concentration-dependent effects on Ca-NS⁺, low concentrations stimulating and high concentrations inhibiting channel activity. 6-Chloropurine cyclic AMP and 8-bromo cyclic AMP produced effects similar to those of cyclic AMP itself. In contrast, 8-[4-chlorophenylthio] cyclic AMP also showed a dual action, but with a high level of activation at all concentrations tested up to 1mm. Ca-NS⁺ channel activity was also predominantly activated by low concentrations of S_p-cAMPS. The activating effects of both S_p-cAMPS and cyclic AMP are antagonized by R_p-cAMPS, which by itself only produced a weak inhibition of Ca-NS⁺ channel activity even at concentrations of 10 μm and above. The results are discussed in terms of a model in which cyclic AMP, and other cyclic nucleotides, modulate the activity of the Ca-NS⁺ channel by binding to two separate sites.     

LTB4 mediated hypoxemia in guinea pigs: relationship to pulmonary and cardiovascular pathophysiology

LTB4 is released in the presence of lung injury and may therefore play a role in the pathophysiology of the lung damage. We therefore, administered LTB4 as an I.V. bolus or as an aerosol to guinea pigs and assessed the physiologic response and the lung histology. After 2 ug of I.V. LTB4 airway pressure (AP) rose transiently by 5 +/- 1 mmHg and at five min was back to baseline while PaO2 fell from 96 +/- 5 mmHg to 78 +/- 3 mmHg and remained low at least 45 min. Static compliance (Cstat) was unchanged. Right ventricular systolic pressure (RVSP) and mean aortic pressure (MAP) rose from 9 +/- 1 to 16 +/- 1 mmHg and 43 +/- 4 to 62 +/- 5 mmHg respectively while cardiac index (C.I.) fell from 266 to 208 ml/kg/min but all values were baseline again by 10 min. Aerosolized LTB4 raised AP by 4.6 +/- 0.2 mmHg while PaO2 fell from 90 +/- 7 to 52 +/- 5 mmHg. AP recovered by 20 min but PaO2 remained low at least for 1 hour. MAP, RVSP and CI and Cstat were unaffected. Both I.V. and inhaled LTB4 increased neutrophil infiltrate in the lung although the water aerosol control did too, preventing us from showing a significant effect with LTB4 aerosol. Indomethacin blocked the airway effects and the hypoxemia after I.V. or aerosolized LTB4 but not the neutrophil infiltrate or the rise in RVSP. It actually enhanced (p less than .05) the rise in MAP after I.V. LTB4. Thus cyclooxygenase released products likely mediated the rise in airway pressure and the prolonged fall in PaO2 after LTB4 in guinea pigs but not the pulmonary and systemic vasoconstriction.