Lipid profiles of Helicobacter pylori colony variants.

A phase variation in Helicobacter pylori has been previously described. In one phase the bacterium had a cell wall lipid content typical for gram-negative bacteria (HpL), whereas in the other phase the bacterium was found to have a cell wall with increased amounts of lysophospholipids (HpS). The conversion is spontaneous, but could also be induced by acid (HpS(ind)) and was associated with in vitro release of Vac A and urease. The purpose of the present study was to determine the full phospholipid content of the cell wall to indicate a molecular mechanism of the colony variation. There were no appreciable differences between the lipid profiles of HpS and HpS(ind), while there were major differences between HpL and the S-variant. In the S-variant, lysophospholipids constituted about 50% of the total phospholipids, as compared to less than 2% in the L-variant. The proportion of total and individual cholesteryl glucosides also showed considerable changes. HpL was dominated by the phosphate-linked cholesteryl glucoside (72%) while the acylated cholesteryl glucoside was the main cholesteryl glucoside of the S-variant (65%). Our results demonstrate a dramatic change in cell wall properties after acid induction and spontaneously in vitro, and suggest some molecular mechanisms for this variation from an in vitro non-virulent to a virulent variant.

The 18 kDa cytochrome c553 from Heliobacterium gestii: gene sequence and characterization of the mature protein.

The 18 kDa cytochrome c553 is the dominant c-type cytochrome in cell membranes of Heliobacterium gestii. After solubilization, this cytochrome was purified in three steps as a complex with two other proteins of 32 and 42 kDa. The redox midpoint potential of the cytochrome c553 was determined to be +215 mV. The EPR spectra clearly show the presence of an ascorbate-reducible low-spin heme with gz = 3.048 and gy = 2.238. The gx = trough could not be detected. In addition, a Cu(II) signal with g = 2.058 was observed, indicating that one component of the cytochrome c553 complex contains a bound copper ion. The gene for the 18 kDa cytochrome c553, cyhA, consists of 429 bp coding for a protein of 142 amino acids. The association of the cytochrome with the cytoplasmic membrane is mediated by two fatty acid molecules, one palmitate and one stearate, that could be identified by mass spectrometry. Both fatty acids are most likely bound to the cysteine residue of the N-terminally processed protein via a glycerol moiety. The amino acid sequence deduced from the DNA sequence exhibits partial identity to the membrane-bound cytochrome c551 from Bacillus PS3 [Fujiwara, Y., Oka, M., Hamamoto, T., and Sone, N. (1993) Biochem. Biophys. Res. Commun. 1144, 213-219] and to the cytochrome c subunit (NorC) of the nitrous reductase from Pseudomonas stutzeri [Zumft, W. G., Braun, C., and Cuypers, H. (1994) Eur. J. Biochem. 219, 481-490].

Colony variation of Helicobacter pylori: pathogenic potential is correlated to cell wall lipid composition.

BACKGROUND: Differences in expression of disease after infection with Helicobacter pylori have so far been connected with host factors and bacterial interstrain variation. In this study, spontaneous and ecology-mediated intrastrain variation was examined. METHODS: Four clinical isolates of H. pylori were shown to give rise to two colony forms. Bacterial morphology was examined by electron microscopy. Bacterial fractions were examined for proteins using ion exchange chromatography and SDS-PAGE; for lipids using thin-layer chromatography, lipid anion-exchange chromatography, column chromatography on silica gel, 31P-NMR, gas chromatography and mass spectrometry. Bacterial in vitro invasiveness and adhesiveness were examined in two different systems, and urease and VacA toxin were assayed by Western blot analysis. RESULTS: H. pylori was shown to give rise to two colony forms: at normal pH the population was dominated by L colonies. One strain was chosen for further studies. Bacteria from L colonies retained VacA toxin and urease, did not invade or adhere to epithelial cells, and contained normal quantities of phosphatidylethanolamine. In a small frequency, spontaneous S colonies were formed. Bacteria from these colonies released VacA and urease, adhered to and invaded epithelial cells and contained increased amounts of lysophosphatidyl ethanolamine and phosphatidyl serine. After addition of HCl to the culture medium (pH6), almost only S colonies were formed. The results demonstrate that environmental factors, such as HCl, can change the bacterial cell wall, and thereby enhance expression of virulence factors of H. pylori in vitro. A similar in vivo variation would have implications for our understanding of the interaction between HCl secretion in the gastric mucosa and H. pylori in the development of peptic ulcer disease.

Up-regulated delta 9-desaturase gene expression by hypolipidemic peroxisome-proliferating fatty acids results in increased oleic acid content in liver and VLDL: accumulation of a delta 9-desaturated metabolite of tetradecylthioacetic acid.

In the liver of rats, monocarboxylic 3-thia fatty acids, tridecylthioacetic acid (C13-S-acetic acid) and tetradecylthioacetic acid (C14-S-acetic acid), increase the mRNA levels of delta 9-desaturase both in a time- and dose-dependent manner. The increased delta 9-desaturase mRNA levels were accompanied by increased delta 9-desaturase activity and increased amounts of oleic acid (18:1 n-9) and delta 9-desaturated C14-S-acetic acid. delta 9-Desaturated C14-S-acetic acid was only detected in phospholipid and cholesterolester species after C14-S-acetic acid treatment. In contrast, C14-S-acetic acid was detected in all the different hepatic lipid fractions, but mainly in the phospholipids. Moreover, C13-S-acetic acid and C14-S-acetic acid were detected in both liver and very low density lipoprotein (VLDL). No delta 9-desaturated 3-thia fatty acid products, however, were found in VLDL. Administration of mono- and dicarboxylic 3-thia fatty acids to rats induced liver expression of the fatty acyl-CoA oxidase gene. After 1 week of C14-S-acetic acid treatment, the levels of fatty acyl-CoA oxidase mRNA increased 5-fold, whereas the delta 9-desaturase mRNA was increased about 1.8-fold. Both fatty acyl-CoA oxidase and delta 9-desaturase mRNA increased about 8-fold after 12 weeks of treatment with C14-S-acetic acid. In conclusion, this study demonstrates that C14-S-acetic acid increases rat delta 9-desaturase gene expression and activity and that changes in hepatic lipids, e.g., 18:1 n-9, are reflected in the VLDL. The peroxisome-proliferating monocarboxylic thia fatty acids are good substrates for desaturases, as delta 9-desaturated metabolites of monocarboxylated thia acids were formed in the liver. Modification of delta 9-desaturation, however, appears not to be related to peroxisome proliferation.

Enhanced hepatic fatty acid oxidation and upregulated carnitine palmitoyltransferase II gene expression by methyl 3-thiaoctadeca-6,9,12,15-tetraenoate in rats.

This study reports the effects of a novel polyunsaturated 3-thia fatty acid, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on serum lipids and key enzymes in hepatic fatty acid metabolism compared to a saturated 3-thia fatty acid, tetradecylthioacetic acid. Palmitic acid treated rats served as controls. Fatty acids were administered by gavage in daily doses of 150 mg/kg body weight for 10 days. The aim of the present study was: (a) To investigate the effect of a polyunsaturated 3-thia fatty acid ester, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on plasma lipids in normolipidemic rats: (b) to verify whether the lipid-lowering effect could be consistent with enhanced fatty acid oxidation: and (c) to study whether decreased activity of esterifying enzymes and diversion to phospholipid synthesis is a concerted mechanism in limiting the availability of free fatty acid as a substrate for hepatic triglyceride formation. Repeated administration of the polyunsaturated 3-thia fatty acid ester for 10 days resulted in a reduction of plasma triglycerides (40%), cholesterol (33%) and phospholipids (20%) compared to controls. Administration of polyunsaturated and saturated 3-thia fatty acids (daily doses of 150 mg/kg body weight) reduced levels of lipids to a similar extent and followed about the same time-course. Both mitochondrial and peroxisomal fatty acid oxidation increased (1.4-fold- and 4.2-fold, respectively) and significantly increased activities of carnitine palmitoyltransferase (CPT) (1.6-fold), 2,4-dienoyl-CoA reductase (1.2-fold) and fatty acyl-CoA oxidase (3.0-fold) were observed in polyunsaturated 3-thia fatty acid treated animals. This was accompanied by increased CPT-II mRNA (1.7-fold). 2,4-dienoyl-CoA reductase mRNA (2.9-fold) and fatty acyl-CoA oxidase mRNA (1.7-fold). Compared to controls, the hepatic triglyceride biosynthesis was retarded as indicated by a decrease in liver triglyceride content (40%). The activities of glycerophosphate acyltransferase, acyl-CoA: 1,2-diacylglycerol acyltransferase and CTP:phosphocholine cytidylyltransferase were increased. The cholesterol lowering effect was accompanied by a reduction in HMG-CoA reductase activity (80%) and acyl-CoA:cholesterol acyltransferase activity (33%). In hepatocytes treated with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate, fatty acid oxidation was increased 1.8-fold compared to controls. The results suggest that treatment with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate reduces plasma triglycerides by a decrease in the availability of fatty acid substrate for triglyceride biosynthesis via enhanced fatty acid oxidation, most likely attributed to the mitochondrial fatty acid oxidation. It is hypothesized that decreased phosphatidate phosphohydrolase activity may be an additive mechanism which contribute whereby 3-thia fatty acids reduce triglyceride formation in the liver. The cholesterol-lowering effect of the polyunsaturated 3-thia fatty acid ester may be due to changes in cholesterol/cholesterol ester synthesis as 60% of this acid was observed in the hepatic cholesterol ester fraction.

Effects of chain length and sulphur position of thia fatty acids on their incorporation into phospholipids in 7800 C1 hepatoma cells and isolated rat hepatocytes, and their effects on fatty acid composition of phospholipids.

Incorporation of thia fatty acids and their effects on the fatty acid composition in phospholipids has been investigated in 7800 C1 hepatoma cells and cultured hepatocytes. 3-Thia fatty acids of chain lengths from dodecyl-to hexadecyl-thioacetic acid were incorporated into phospholipids during a 3-day incubation. Longer and shorter 3-thia fatty acids were barely detectable. Tetradecylthioacetic acid, 3-thia stearate, and their delta9- desaturated derivatives were maximally incorporated into whole-cell phospholipids. The amount of tetradecylthioacetic acid incorporated into phospholipids of hepatoma cells remained almost identical in cells cultured for 3 days or adapted over a period of 1 year. Delta9-desaturated metabolites of long chain thia fatty acids (C13-to C16-S-acetic acid) were identified by GC-MS in phospholipids. 3-Thia stearate appeared to be the best substrated for delta9 desaturase. Incubation of hepatoma cells with thia fatty acids led to alterations in the amount of normal fatty acids in total phospholipids. The amounts of 16:0 and 18:1 decreased and 18:2 (n-6) and 20:5 (n-3) increased. Changes in the normal fatty acid composition of phospholipids were seen both with thia acids incorporated into phospholipids and those not incorporated. These effects, therefore, may be only partially dependent on displacement of normal fatty acids by thia fatty acids. Morris 7800 C1 hepatoma cell acyl-CoA synthetase (ACS) and peroxisomal acyl-CpA oxidase (ACO) were induced by thia fatty acids of all chain lengths, and with the sulphur atom(s) in different positions. Control experiments with hepatocytes revealed a similar incorporation of thia fatty acids in these physiologically more normal cells.

Acute modulation of rat hepatic lipid metabolism by sulphur-substituted fatty acid analogues.

A single oral dose of two 3-thia (3-thiadicarboxylic and tetradecylthioacetic acids) and of 4-thia (tetradecylthiopropionic acid) fatty acids were administered to normolipidemic rats and their effects on lipid metabolism over a 24 hr period were studied. All three thia fatty acids could be detected in plasma 2 hr after treatment. Tetradecylthioacetic and tetradecylthiopropionic acids were detected in different hepatic lipid fractions but were incorporated mainly into hepatic phospholipids. Two hours after administration hepatic mitochondrial beta-oxidation and the total liver level of long-chain fatty acyl-CoA increased with a concomitant decrease in saturated fatty acids, total hepatic malonyl-CoA and plasma triacylglycerol levels in the 3-thia fatty acid groups. Tetradecylthiopropionic acid administration caused a decrease in mitochondrial beta-oxidation and an increase in plasma triacylglycerol at 24 hr. The activities of key lipogenic enzymes were unaffected in all treatment groups. Plasma cholesterol level was reduced only at 8 hr in 3-thiadicarboxylic acid treated rats although 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase was suppressed already at 2, 4, 8 and 12 hr. The results show that thia fatty acids are rapidly absorbed and are systemically available after oral administration but the 3-thia fatty acids reached systemic circulation more slowly and less completely than the 4-thia fatty acid. Very low levels of the thia fatty acids are detected in plasma 24 hr after a single administration. They are incorporated into all hepatic lipid classes, especially phospholipids. Rapid incorporation of a non beta-oxidizable thia fatty acid into hepatic lipids may cause a diversion of other fatty acids from glycerolipid biosynthesis to mitochondrial beta-oxidation. Stimulation of mitochondrial beta-oxidation and suppression of HMG-CoA reductase are primary events, occurring within hours, after 3-thia fatty acid administration. The hypotriglyceridemic effect of the 3-thia fatty acids observed at 2-4 hr is independent of the activities of key lipogenic and triacylglycerol synthesising enzymes.

Serum lipids in children with xeroderma from an inland district of Sri Lanka.

Children living on plantations in inland districts of the southeastern part of Sri Lanka frequently develop a skin condition on the legs described as mosaic skin or xeroderma. This condition is characterized by atrophic, dry, shining and scaly skin. The etiology is unknown. A food frequency survey indicated a low energy intake, a diet with a low fat content, and anthropometric data have shown a high prevalence of malnutrition within this group. The skin condition brought attention to a possible deficiency of essential nutrients, especially essential fatty acids. In order to investigate the possible association with a deficiency of essential fatty acids, blood samples were collected from both children having signs of xeroderma and controls. The total amount of phospholipids was low, but the fatty acid profile of this lipid class was similar to the controls. A vitamin A deficiency was indicated by low levels of its transport proteins. A multifactorial etiology where vitamin A deficiency may play a role is discussed.

The regulation of fatty acid chain elongation in rat liver microsomes: role of fasting and CoASH.

The elongation system for palmitic acid in rat liver microsomes was decreased to 1/3 by fasting, while the elongation of eicosapentaenoic acid was not sensitive to fasting. The rate of eicosapentaenoic acid elongation in the fed state was 50% higher than using palmitic acid as a substrate. The saturated and polyunsaturated fatty acyl-CoA substrates exhibited positive cooperativity on the rate-limiting condensing step in the elongation system, with a Hill constant of approx. 2. An inhibition by CoASH on the total elongation reaction as well as on the condensation step was demonstrated using acyl-CoA substrates, and followed a hyperbolic pattern. The concentrations giving a 50% inhibition (30-70 microM) were in the range found in rat hepatocyte cytosol, indicating that free CoASH has the potential to act as a physiological regulator.

Gas chromatographic measurement of 3- and 4-thia fatty acids incorporated into various classes of rat liver lipids during feeding experiments.

A practical procedure is described for the quantitative measurement of the amount of acyl units derived from tetradecylthioacetic acid (effecting hypolipemia in rats) and tetradecylthiopropionic acid (effecting hyperlipidemia). The procedure involves three main successive steps: (1) extraction; (2) solid-phase lipid class separation yielding free fatty acids, phospholipids, triacylglycerides, cholesterol esters, and diacylglycerides without crosscontamination; and (3) gas chromatography of hydrolyzed lipids derivatized to picolinyl esters, combined with unambiguous identification by gas chromatography-mass spectrometry. The overall recoveries of heptadecanoyl lipids added as internal standards during extraction were 94-96%, except for cholesteryl heptadecanoate where the recovery was 60% owing to incomplete hydrolysis. Recoveries of thia fatty acids from samples spiked with these compounds were 95%. Flame-ionization response factors were found to be 0.92 and 0.81 for the tetradecylthioacetic acid and tetradecylthiopropionic acid picolinyl esters, respectively, compared to that of heptadecanoic acid. The lower limit of quantitation was 25 pmol as injected. Measurement of the amount of thia fatty acyl units in rat plasma and in liver lipids 4 h after administration of single doses by gastric intubation indicated efficient absorbtion and rapid incorporation into liver lipids, particularly in the phospholipid fraction. Both plasma clearance and channelling into lipids was slower for the 4-thia fatty acid.

Hormonal and substrate regulation of 3-thia fatty acid metabolism in Morris 7800 C1 hepatoma cells.

The 3-thia fatty acid tetradecylthioacetic acid (TTA) has recently been shown to inhibit growth rate and increase peroxisomal acyl-CoA oxidase (ACO) (EC 1.3.99.3) activity in the Morris 7800 C1 hepatoma cells. Dexamethasone potentiates and insulin antagonizes these effects of TTA. We demonstrate here the metabolism of the 3-thia acids in these cells and the influence of insulin and dexamethasone on this. (1) The Morris 7800 C1 hepatoma cells exhibited a low omega-hydroxylation activity of the 3-thia acid (and lauric acid). The combination of TTA and dexamethasone induced the omega-hydroxylation and ACO activities in these cells. TTA alone induced ACO activity, but not omega-hydroxylation activity. Insulin counteracted the induction of both enzyme activities. These results indicate that these two enzyme activities are under similar but independent regulation. (2) Hepatoma cells grown with 80 microM TTA in the medium accumulated phospholipids containing the 3-thia fatty acid. After 7 days, TTA accounted for approx. 40% of the total fatty acids in the phospholipids. In addition, TTA affected the incorporation of endogenous fatty acids into phospholipids by decreasing the amounts of palmitic (C16:0) and vaccenic (C18:1(n-7)) acid and increasing the amounts of linoleic (C18:2(n-6)) and alpha-linolenic (C18:3(n-3)) acid in the phospholipids. (3) Dexamethasone increased the incorporation of labelled TTA into both phospholipids and triacylglycerol. Most of the labelled triacylglycerol formed was secreted into the medium. Insulin increased the incorporation of labelled TTA into triacylglycerol, but not into phospholipids. The labelled triacylglycerol formed was retained in the cells.

Opposite effect of cold on energetic efficiency in normal and obese Wistar rats with hypothalamic lesions.

Reduced thermogenesis in brown adipose tissue (BAT) may contribute to increased energetic efficiency and obesity in rats with ventromedial hypothalamic (VMH) lesions. Thermogenic activity of BAT is a function of the environmental temperature. If a relationship exists, it follows that the increased energetic efficiency of VMH-lesioned rats likewise should be governed by temperature. We have therefore investigated the energy balance of normal and VMH-lesioned rats housed at 30 degrees C and 10 degrees C. Experiments at differing feeding levels allowed calculation of maintenance energy requirements and the net energetic efficiencies of each group. VMH-lesioned rats at thermoneutrality (30 degrees C) accumulated more body fat at all feeding levels than did normal rats. Maintenance energy requirement was reduced, but the net energetic efficiency did not differ significantly from normal. The reduced maintenance energy requirement of lesioned rats persisted at 10 degrees C. Net energetic efficiency decreased in normal rats acclimated to cold but increased in the lesioned group. The difference was significant (P less than 0.05). The cold-induced increase in interscapular brown adipose tissue (IBAT) oxidative capacity of VMH-lesioned rats was only half that of normal rats. Differences in BAT thermogenesis may be the basis for the differing temperature effects on net energetic efficiency.

Oxidative capacity of tissues contributing to thermogenesis in eider (Somateria mollissima) ducklings: changes associated with hatching.

1. The development of liver and skeletal muscle oxidative capacities during hatching of the common eider (Somateria mollissima) in the Arctic has been investigated by monitoring tissue cytochrome c oxidase activity. 2. The specific activity of the liver enzyme did not change as the embryo underwent hatching, nor during subsequent growth of the duckling into adulthood. 3. Thigh muscle enzyme specific activity increased by a factor of 3.4 during the 24 h prehatching period, remained elevated for at least 48 h after hatching, and then returned to the embryonic (-24 h) level in adults. 4. Histochemically visualized NADH-tetrazolium reductase of a typical red thigh muscle, M. vastus lateralis, showed a distinct increase in activity as the hatching process progressed to completion. 5. Electron microscopy of sectioned M. vastus lateralis revealed a dramatic increase in the density of the myofibrillar structure (number of mitochondrial profiles per unit area), and in the surface area of mitochondrial crista membranes in the course of the 48 h interval from 1 day prehatching to 1 day after hatching. 6. The significance of these changes for the scaling of thermoregulatory heat generation in the newly hatched eider duckling is discussed.

Modes of thermal protection in newborn muskoxen (Ovibos moschatus).

The muskoxen (Ovibos moschatus), a native of Greenland and the Canadian North West Territories, give birth in late April, and the newborn calves are known to tolerate an ambient temperature (Ta) of -35 degrees C. At birth the calves weigh about 8 kg, increasing in weight with 0.6 kg . day-1 for the first 30 days. With a deep body temperature (DBT) of 39.5 degrees C (range 37.7-41.3 degrees C) the newborn calves are consequently able to maintain a thermogradient of at least 70 degrees C between body core and the environment. The calves use primarily two modes of thermal protection: High metabolic heat production and prime fur insulation. Metabolic rate was about 3.5 W . kg-1 at thermoneutrality in calves aged from 8 h to 7 days. Lower critical temperature at this age was about -7 degrees C and a drop in Ta to -30 degrees C increased metabolism to about 5.3 W . kg-1. Upper critical temperature at age 4-7 days is as low as 20 degrees C, while it in calves aged only 18-24 h appears to be even lower. The calves possess great amounts of brown adipose tissue (BAT) at birth. Mitochondria from the BAT deposits were isolated and found to be in an extremely loose-coupled state with a great capacity for thermogenesis. Skeletal muscle contained very few mitochondria and is hardly employed in aerobic non-shivering thermogenesis. Calves shiver visibly while drying just after birth, but are normally not seen shivering thereafter. The conductance value for the dry pelt of newborn calves averaged 3.2 W . m-2 . 0 degrees C-1 (n = 4). Wetting of the pelt with ice-water at a Ta of 3 degrees C increased conductance to 8.8 W . m-2 . 0 degrees C-1. The conductance of the pelt was also influenced by wind, being 10 W . m-2 . C-1 at a wind-speed of 10 m . sec-1. The legs of the newborn calves are heavily furred and countercurrent circulation is not present, subcutaneous temperature just above the hooves being +29.8 degrees C at Ta of -24 degrees C as compared to 37.5 degrees C on the back. The newborn calves could cope with a Ta of -30 degrees C without apparent problems under experimental conditions, but they suffered hypothermia when exposed to a Ta of -33 degrees C in combination with wind of 10 m . sec-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Vitamin C deficiency in growing willow ptarmigan (Lagopus lagopus lagopus).

Willow ptarmigan chicks raised on a diet containing 265 mg ascorbic acid/kg develop scury-like symptoms and die by 4 weeks of age. If blueberry plants are given as an ad libitum supplement to this diet, the malady is prevented. We have described the clinical, pathological and histological changes which accompany this malnutrition and conclude that they are in accord with the description of scurvy in guinea pig and man. Biochemical determination of ascorbic acid synthesis in the kidney of ptarmigan chicks indicated a rate of synthesis five times that found in livers of growing white rats. Blueberry plants and many other plants found in the natural diet of ptarmigan chicks contain 2,000 to 5,000 mg ascorbic acid/kg dry weight. Feeding experiments showed that the pathological signs were avoided and that already afflicted chicks recovered if the vitamin C content of the diet was raised to 750 mg/kg dry weight of food. Since the food intake of the chicks was 5 to 8 g/day the daily requirement of external vitamin C is about 150 mg/kg body weight. To our knowledge this is the first example of an animal which, while producing vitamin C itself, requires substantial amounts of external vitamin C to survive.

Newborn northern fur seals (Callorhinus ursinus)--do they suffer from cold?

Several hundred thousand northern fur seals (C. ursinus) are born each summer during July at St. Paul Island in the Bering Sea. The weather in the area is usually cold, wet, and windy during the breeding season. At birth the pups are small (5--6 kg) and insulated only by a partly wettable pelt and a 2- to 4-mm layer of blubber. In air, the pups' lower critical temperature appears to be below the 6 degrees C 50-yr record low July temperature for the islands. During rainy weather much of the insulative value of the pelt is lost, and the pups, which already have a high resting metabolic rate of 3.5 W.kg-1, must increase heat production by shivering and/or nonshivering thermogenesis to maintain deep body temperature. The high level of metabolism (up to 18 W.kg-1) is supported by a very rich milk. The pups will, nevertheless, become hypothermic if their insulation is not improved through peripheral vasoconstriction and shedding of water from the pelt by periodic shudder. Even with these protections the newborn and very young pups are brought close to their limit of tolerance during rainy and windy days. Unfit pups are likely to succumb under such circumstances.

A source of nonshivering thermogenesis in fur seal skeletal muscle.

The mitochondria from the subscapular muscle of naturally cold-stressed 10- to 15-year-old northern fur seals (Callorhinus ursinus) were loosely coupled upon isolation, whereas the mitochondria from the same muscle of warm-acclimated pups of the same age were tightly coupled. Thus, loose-coupled muscle mitochondria might provide an important vehicle for nonshivering thermogenesis in this species.

Some aspects of temperature regulation in newborn harp seal pups.

Harp seals are born on the drifting ice of the North Atlantic Ocean during arctic winter when temperatures of -20 degrees C, occasionally in combination with wind of 10 m/s, might prevail for days. At birth the pups lack subcutaneous blubber and the wet infantile fur has a conductance value of 30.0 W . m-2 . degrees C-1, as compared with only 2.0 W . m-2 . degrees C-1 when dry. While still wet immediately after birth the pups are nevertheless able to retain body core temperature by shivering. This activity leads to reduction of muscle fat and glycogen stores. Nonshivering thermogenesis commences in thermogenic adipose tissue by virtue of loosely coupled mitochondria. Thermogenic adipose tissue is found at birth both as a subcutaneous layer along the back and as internal deposits around venous plexuses in the neck, on the pericardium, on the kidneys, and the abdominal walls. After about 3 days of suckling the subcutaneous adipose tissue loses its thermogenic function being gradually transformed into blubber, whereas the internal deposits persist at least until the pups venture into water at the age of 3-4 wk.

Brown adipose tissue-a factor in the survival of harp seal pups.

The respiratory characteristics of mitochondria isolated from the subcutaneous brown adipose tissue of newborn harp seals indicate that the tissue is thermogenically active. Temperature recordings in vivo revealed, in fact, that the tissue was maintained at a temperature close to that of the body core during immersion of the pups in ice-water. Beta-adrenergic blockade markedly increased the cooling rates at both locations in ice-water, while curarization, accompanied by artificial respiration did not. We conclude that nonshivering thermogenesis through activated brown adipose tissue plays a decisive role in the defence against cold in the newborn harp seal.

Differential effects of temperature on activity of LDH from seal and sheep skin.

The homeothermic seal possesses a heterothermic skin, while the skin of the sheep behaves as a truly homeothermic tissue. A new method for skin homogenization is described. The effect of temperature on the catalytic behaviour of seal and sheep skin LDH has been investigated by fluorimetric activity measurements, with results presented as Arrhenius plots. The seal skin enzyme had ten times higher activity at low temperatures as compared to the sheep skin. The mechanisms responsible for this different behaviour are discussed.