Regulation of ANP secretion in insulin-dependent diabetes mellitus and the influence of autonomic neuropathy.

In order to determine the effect of diabetic autonomic neuropathy (DAN) on the atrial natriuretic peptide (ANP) response to dynamic stimuli, we studied the ANP response to 60 degrees head-up and 60 degrees leg-up tilt in diabetic subjects with (DAN + ve, n = 8) and without (DAN - ve, n = 8) evidence of autonomic neuropathy and seven matched non-diabetic controls. Mean baseline plasma ANP concentrations were similar in all three groups. Head-up tilt was associated with a fall in plasma ANP in all seven healthy controls (21.8 (16.8-30.7) to 16.8 (7.1-29.1), P = 0.06, mean (range)), seven of the eight DAN - ve (16.9 (6.5-33.7) to 8.5 (3.0-21.1), P = 0.015) and all eight DAN + ve subjects (27.3 (8.5-101.5) to 15.4 (1.0-67.6), P = 0.044). Leg-up tilt caused a rise in plasma ANP in six of the seven healthy controls (17.6 (7.5-27.9) to 22.4 (15.2-48.1), P = 0.041), six of the eight DAN - ve (12.5 (7.8-27.8) to 15.5 (7.3-31.3), P = 0.054) and seven of the eight DAN + ve subjects (18.2 (2.8-55.1) to 25.1 (4.5-92.8), P = 0.013). There was no significant difference in the fall in plasma ANP during head-up tilt or in the rise in plasma ANP during leg-up tilt between the three groups. We conclude that the regulation of ANP secretion is normal in diabetes mellitus, and is unaffected by the presence of autonomic neuropathy.     

Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.     

Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells

During growth, Dictyostelium cells continuously secrete a factor, PSF, that accumulates in proportion to cell density. At sufficient concentration, it triggers the production of discoidin I and certain lysosomal enzymes. Our earlier studies demonstrated these effects of PSF on protein and enzyme levels [Clarke et al., Differentiation 34:79-87, 1987; Clarke et al., Dev Genet 9: 315-326, 1988]. In the present study, we have examined whether PSF induces increased mRNA levels. By Northern blot analysis, we have found that discoidin I mRNA accumulates in exponentially growing NC4 cells as the cells reach high density; significant levels of mRNA are detectable in cells growing either on plates or in suspension, beginning about four generations before the end of exponential growth. High levels of discoidin I mRNA are also found in low-density cells grown in the presence of buffer conditioned by high-density cells. These results indicate that PSF induces the accumulation of discoidin I mRNA. Other "early developmental" genes, pCZ22 and the early I genes (16, 18, and 111), are also expressed in exponentially growing cells at high density or in the presence of conditioned buffer. We conclude that several genes previously found to be preferentially expressed very early in development are actually induced during late exponential growth by PSF.     

Diazinon resistance, fluctuating asymmetry and fitness in the Australian sheep blowfly, lucilia cuprina

Genetic evidence suggests that the evolution of resistance to the insecticide diazinon in Lucilia cuprina initially produced an increase in asymmetry. At that time resistant flies were presumed to be at a selective disadvantage in the absence of diazinon. Subsequent evolution in natural populations selected modifiers to ameliorate these effects. The fitness and fluctuating asymmetry levels of resistant flies are currently similar to those of susceptibles. Previous genetic analyses have shown the fitness modifier to co-segregate with the region of chromosome III marked by the white eyes, w, locus, unlinked to the diazinon resistance locus, Rop-1, on chromosome IV. This study maps the asymmetry modifier to the same region, shows, as in the case of the fitness modifier, its effect to be dominant and presents data consistent with the fitness/asymmetry modifier being the same gene (gene complex). These results suggest changes in fluctuating asymmetry reflect changes in fitness.     

Platelet-activating factor induces the expression of early pregnancy factor activity in female mice

When synthetic platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was injected into mature female mice during dioestrus, pro-oestrus or oestrus, it induced the expression of early pregnancy factor (EPF) activity in the sera of these animals within 1 h of injection. The sera of similarly injected males, metoestrous or immature females did not display any EPF activity. The results suggest that embryo-derived PAF may be the ovum factor responsible for triggering the generation of serum EPF activity during the preimplantation stages of pregnancy.     

Gonadal development and gonadotrophin secretion in the male vole (Microtus agrestis) after an abrupt change in photoperiod

Male voles were reared from birth to age 28 days in 6L:18D. Pairs of animals showing similar sexual development were assigned at random to 16L:8D or 6L:18D. Treatments continued for a further 56 days. Increase in the activity of the hypothalamo-hypophysial system occurred within 4 days of exposure to 16L:8D, as shown by significant elevation of plasma LH and FSH. Pituitary LH did not increase until Day 7, and pituitary FSH did not increase until Day 21. After exposure to 16L:8D for 4 days, pituitary FSH was lower than in corresponding animals in 6L:18D. These discrepancies between pituitary and plasma values of gonadotrophins indicate that increase in hormone release occurs before synthesis is fully stimulated. Enhanced output of testicular hormones probably began between Day 7 and Day 14, as indicated by an increase in seminal vesicle weight, yet plasma and pituitary concentrations of LH and FSH remained elevated. This suggests that long photoperiods may cause direct stimulation of the hypothalamo-hypophysial system which increasing values of testicular hormones are initially unable to inhibit. The response of this system in voles to an abrupt change from a non-stimulating to a stimulating photoperiod has a time course resembling that for the Soay ram but appreciably slower than for the Japanese quail.     

Uptake and metabolism of lactosylceramide on low density lipoproteins in cultured proximal tubular cells from normal and familial hypercholesterolemic homozygotes

The metabolism of low density lipoproteins (LDL), and LDL modified by reductive methylation (M-LDL) of lysine residues, was studied in proximal tubular (PT) cells both from normal human kidney and from urine of patients with homozygous (LDL receptor-negative) familial hypercholesterolemia (FH). LDL and M-LDL was labeled either in the protein moiety with 125I or in the lactosylceramide moiety with 3H. The binding and degradation of 125I-LDL in normal cells was saturable and displaced by unlabeled LDL but not by M-LDL. The uptake of [3H]lactosylceramide (LacCer) low density lipoprotein in normal renal cells was saturable, and time and temperature-dependent. Exogenously derived [3H]LacCer on LDL was rapidly taken up and catabolized to monoglycosylceramide, or it was utilized for the endogenous synthesis of globotriaosylceramide (trihexosylceramide) and globotetraosylceramide (tetraglycosylceramide). [3H]LacCer M-LDL was taken up less avidly and metabolized less extensively than [3H]LacCer-LDL in normal cells. In homozygous FH renal cells the binding of 125I-LDL was not saturable and not displaced by unlabeled LDL. 125I-LDL degradation did not occur in FH cells. The homozygous FH PT cells took up a 2-fold greater amount of exogenously derived [3H]LacCer on LDL than normal cells. Yet, most of the [3H]LacCer taken up by FH PT cells accumulated as LacCer, and only small amounts were metabolized to monoglycosylceramide, globotriaosylceramide (trihexosylceramide), or globotetraosylceramide (tetraglycosylceramide). When normal and FH PT cells were preincubated with LDL (0-100 micrograms/ml medium), there was a 5-fold increase in cellular LacCer levels in FH cells at saturating levels of LDL, whereas there was about a 50% decrease in LacCer levels in normal cells. While the high affinity binding of LDL was not essential for the delivery of LacCer to cells, the data support the conclusion that LDL binding to the LDL receptor facilitates further LacCer processing and metabolism in normal renal cells. We speculate that [3H] LacCer is taken up by FH homozygous cells via a LDL receptor-independent mechanism and accumulates in the cells without significant metabolism. LacCer taken up by this mechanism contributes to the storage of LacCer in FH PT cells.     

Seasonal changes in LH secretion in normal ewes and ewes which grazed oestrogenic clover

Plasma luteinizing hormone (LH) concentrations were measured in normal (control) Corriedale X Merino (comeback) ewes and in clover-infertile comeback ewes which had grazed oestrogenic Yarloop clover (Trifolium subterraneum L. cv. Yarloop) for more than 4 years. Plasma LH concentrations were measured in samples taken at 20-min intervals for 6 h during the dioestrous stage of the oestrous cycle in the breeding season (BS) and during the anoestrous season (AS). In the control ewes during BS, transitory elevation in plasma LH concentration (pulses) occurred, reflecting secretory episodes, with a frequency of one per 5.2 h. This frequency fell to one per 16.5 h during the anoestrous season. In clover-infertile ewes, LH pulses occurred with a frequency of one per 4.5 h during BS and one per 4.9 h during AS (difference not significant). In the controls, plasma LH levels were higher (P less than 0.05) during BS (mean +/- s.d. = 1.2 +/- 0.4 ng/ml, n = 9) than in AS (0.7 +/- 0.3 ng/ml, n = 5). In the clover-infertile ewes, plasma LH levels in BS (1.3 +/- 0.6 ng/ml, n = 12) were similar to those of controls. During AS, plasma LH levels in the clover-infertile ewes (1.0 +/- 0.6 ng/ml, n = 10) remained similar to their BS levels, being significantly (P less than 0.05) higher than LH levels in the controls at this time. These studies indicate that the higher plasma concentrations of LH which have been reported in clover-infertile ewes arise from more frequent LH pulses. Furthermore, in contrast to normal ewes, average plasma LH, reflecting pulse frequency, is not reduced in AS. This supports the view that ingestion of phytooestrogens affects neural centres involved in regulating LH secretion.