Morphofunctional changes in the small intestine epithelium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis> in the course of hibernation

The structure and function of small intestine epithelium have been studied in overwintering frogs at various stages of hibernation. Experiments on the in vitro absorption of arginine vasotocin (AVT) have shown that the epithelium ability for absorption of this nonapeptide without hydrolysis persists during hibernation. However, there is a decline in AVT absorption observed in January–February and later on (as compared with that in October–December) which is most pronounced in March–April. Changes in the epithelial structure appear by midwinter and are growing towards spring. In April–May, enterocyte height decreased by 33%, microvilli length by 40%, and number of mitochondria by 57% relative to the values at the beginning of hibernation. The absence of signs of breakdown attests to the adaptive nature of the observed changes. The dynamics of the studied parameters reveals morphological and functional plasticity of small intestinal epithelium in Rana temporaria over the hibernation period.     

A Novel Type of Substrate Specificity of Rice BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase) with Mixed Endopeptidase and Carboxypeptidase

The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984)     

Metabolism of Carbon-14 Labelled Arginine, Citrulline and Ornithine in Intact Apple Stems

A study was made of the absorption and metabolism of arginine-guanido-¹⁴C, citrulline-carbamyl-¹⁴C and ornithine 1-¹⁴C which had been applied to apple stem internodes by a perfusion technique at intervals throughout the year. The results showed that these amino acids were interconverted according to the Krebs-Henseleit cycle always in the direction arginine-ornithine-citrulline-arginine. In addition ornithine was degraded by another pathway with loss of CO₂. This loss was particularly extensive during the period June to August when labelled proline and glutamic acid were also found. The evidence is consistent with the initial breakdown of ornithine taking place via glutamic semi-aldehyde.     

Seasonal pattern of LH and testosterone secretion in adult male fallow deer, Dama dama

At monthly intervals during the year blood samples were collected every 20 min for 12 h from 4 entire and 2 prepubertally castrated adult fallow deer bucks. In the entire bucks there were seasonal changes in mean concentrations and pulse frequencies of plasma LH. Mean concentrations in late summer and autumn were 3-6 times higher than during other seasons. LH pulse frequency was low (0-1 pulses/12 h) during most of the year and increased only during the 2-month period (January and February) that marked the transition from the non-breeding season to the autumn rut. During this period there was a close temporal relationship between pulses of LH and testosterone. However, during the rutting period (March and April) episodic secretion of testosterone, manifest as surges in plasma concentrations of 4-6 h duration, was not associated with any detectable pulses in LH although mean plasma concentrations of LH remained elevated. During the rut, the surges of plasma testosterone occurred at similar times of the day. Plasma profiles in May indicated very low concentrations of LH and testosterone secretion in the immediate post-rut period. Castrated bucks exhibited highly seasonal patterns of LH secretion, with mean plasma LH concentrations and LH pulse frequency being lowest in November (early summer) and highest in February and March (late summer-early autumn). Mean concentrations and pulse frequency of LH in castrated bucks were higher than for entire bucks at all times of the year.     

Changes in the rate of growth in a stunted roach Rutilu rutilus population

Data is presented concerning the growth of roach in a South Lancashire lake. Results from a sample of roach in May 1969 indicate the slowest growth rate for which published records are available. A comparison of the length/age and weight/age relationships between data for May 1969 and May/June 1971 yields significant between-year differences indicating an increase in the growth rate during that period. A study of the growth history of individual year classes using back-calculated values from operculum measurements suggests that for both sexes the observed increase in the rate of growth commenced after the 1965–67 period during which very weak year classes were produced. The mean length of females appears to have been larger than that of males from the age of four onwards but the 95% confidence intervals are almost totally overlapping for each pair of values and a statistical comparison of growth rates between the sexes indicates no significant differences. Data for the sexes combined is compared with published results from other British and Continental waters.     

Experimental Studies on the Formation of Volatile Nitrogen Compounds Induced by Pseudomonas Fragi in A Synthetic Medium with Amino Acids as Source of Nitrogen

The influence of the growth of Pseudomonas fragi strain F 111 was studied in a synthetic medium. It was shown that volatile nitrogen compounds were rapidly liberated as a result of the decomposition at 5°C of arginine and histidine. A later and slower increase of TVN was observed in media, which contained lysine or urea. From the other 7 amino acids which were included in the test, no increase of TVN was observed to be caused by the strain F 11. It was shown that within the range of 0.05 to 0.8 % histidine added to the basic salt solution the amount of TVN liberated was correlated to the amount of histidine included in the media. At the TVN maximum approx. 50 % of the amino acid nitrogen of the medium was recovered as TVN. It was further shown that the liberation of TVN was correlated to the added amount of arginine and histidine included in the growth medium. The presence of lysine also influenced on the TVN maximum which was reached. In the synthetic medium to which arginine or histidine was added the growth of strain F 111 was stimulated by the presence of glucose. The acids produced by the oxidative breakdown of glucose were neutralized partly by the volatile bases produced in the decomposition of amino acids. The ability of the Pseudomonas fragi to grow in anaerobic conditions and to produce enzymes which could decompose histidine was studied. It was concluded that the anaerobic breakdown of histidine was similar to the anaerobic breakdown of arginine reported by other authors.     

Trends in mucosal immunity in Antarctica during six Australian winter expeditions

The mucosal immune status of Australian Antarctic personnel was monitored during six wintering expeditions at two Australian Antarctic Research Stations, Casey in 1992, 1993, 1994, and Mawson in 1992, 1995, 1996. Salivary immunoglobulin and albumin levels were examined for differences between stations and expeditions, and for monthly changes over the expedition year. Salivary IgA and IgM concentrations were on average higher for the 1993 Casey expeditioners, and all salivary protein levels were lower for 1996 Mawson expeditioners compared to levels of the other expeditions. The change in salivary IgA and IgM concentrations over the 1-year period revealed a consistent pattern between expeditions. Salivary IgA levels were lower in March, April and May compared to other months of the year (P = 0.0002). Salivary IgM levels were lowest in the first 4 months of the year, with peak levels in June and July (P < 0.0001). There were no changes in salivary IgG and albumin concentrations over the expedition year. Though the cause of the changes in salivary IgA and IgM levels over the year is unknown, the changes could reflect alterations in mucosal immunity in response to stressors associated with isolation.     

Variation in laying date in relation to spring temperature in three species of tits (Paridae) and pied flycatchers Ficedula hypoleuca in southernmost Sweden

This study documents the advancement of laying dates in three species of tits (Paridae) in southernmost Sweden during recent decades, and the absence of a similar response in the pied flycatcher Ficedula hypoleuca. It is based on several different nestbox studies; the oldest one starting in 1969. During 1969 to 2012, mean spring temperatures in the study area increased by between 0.06 and 0.08°C per year, depending on the period considered. Great tits Parus major, blue tits Cyanistes caeruleus and marsh tits Poecile palustris, which generally start egg laying between the last week of April and the first week of May, all advanced laying date at a similar rate during the study period (0.25 d yr^–1). This indicates that these species were similarly affected by increasing temperatures. When accounting for mean spring temperature variation, we still found an advancement of laying date over the study period, mostly due to such relationships among marsh and blue tits. This result could reflect ongoing microevolution favouring earlier laying, but could also be a result of other factors such as increased intra‐ or inter‐specific competition for early breeding. Pied flycatchers, which generally lay during the third week of May, did not significantly advance the date of egg laying despite that the long‐term trend in the increase in ambient temperature during the 30‐d period preceding the start of egg laying was similar for pied flycatchers compared to the tit species.     

Regulation of arginine and proline catabolism in Bacillus licheniformis

The enzymes in the arginine breakdown pathway (arginase, ornithine-delta-transaminase, and Delta'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Delta'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Delta'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.     

Seasonal differences in plasma testosterone profiles in buffalo bulls

Testosterone was measured by radioimmunoassay in blood samples collected hourly over 10 h from two adult buffalo bulls in April, May, August and December. The basal concentrations were below 0.2 ng/ml while peak concentrations ranged from 0.35 to 1.65 ng/ml, with not more than one complete peak occurring during a 10 h period. Both bulls had similar testosterone profiles within each sampling period but differences were evident between periods, the mean concentration being highest in August and falling through December and April to the lowest levels in May. Testosterone concentrations in buffaloes are therefore lower than those in other domestic species, and appear to vary during different times of the year.     

Uptake and Distribution of Nitrogen in Young Apple Trees after Application of Nitrate or Ammonium, with Special Reference to Asparagine and Arginine

Apple rootstocks M.7 were given either nitrate or ammonium at the end of September. Until the following June, total and protein nitrogen and the composition of the soluble nitrogen fraction were followed in the roots, stem-bark, and new growth. Nitrogen from both sources was readily absorbed in roughly the same amounts. Absorption occurred in the autumn and especially in the following spring. Incorporation of the absorbed nitrogen took place exclusively in the young roots. Arginine and, to a lower degree, asparagine were by far the most abundant of the soluble amino compounds and reached levels far above those in the unfertilized trees. The asparagine/arginine ratio was rather high during the first few weeks after fertilization but then dropped steadily to a low winter level. During May and June there was a gradual decrease to very low levels, especially in the new growth. The data suggest that the absorbed nitrogen is incorporated into asparagine and that arginine is formed from asparagine if the supply of nitrogen exceeds the immediate needs. Throughout the experimental period, ammonium fertilization led to higher values of the asparagine/arginine ratio than did nitrate nutrition. This is explained in terms of an enhanced production of asparagine and a retarded conversion into arginine. It is argued that asparagine is the main translocation compound for nitrogen. The possibility is discussed that, in addition, arginine moves upwards by a process of exchange along the negatively charged walls of the xylem vessels.     

Temporal expression of different pathways of 1-arginine metabolism in healing wounds

Arginine can be metabolized by inflammatory cells through at least two pathways. One is an oxidative l-arginine deiminase (OAD) that results in the formation of citrulline and reactive nitrogen intermediates. The other is arginase, which determines the production of ornithine and urea. The temporal expression of these pathways in an experimental wound model (s.c. implanted polyvinyl alcohol sponges in the rat) was investigated by examining the concentrations of amino acids and of nitrite in fluids obtained from the sponges 6 h to 15 day after implantation. These analyses revealed two distinct periods during which the arginine concentration in the fluids was markedly below plasma levels. During the early period (less than 3 days after sponge implantation) wound fluid contained more citrulline and nitrite than at any other time, suggesting OAD activity. In contrast, ornithine accumulated in the fluids during the late decrease in arginine concentration that extended beyond day 3, during which time the wound fluid also contained a high arginase activity. This time-dependent expression of different pathways of arginine metabolism in wounds was confirmed in sponge cultures containing [guanido-14C]-l-arginine. Cells contained in sponges harvested less than 48 h after implantation metabolized labeled arginine mainly to labeled citrulline, whereas labeled urea was produced during culture of sponges harvested after this time. The low arginine content of wound fluid did not appear to be rate limiting for the expression of OAD in late sponges because no OAD activity was evidenced when 4 mM arginine was added to the cultures. These results indicate that the OAD pathway is expressed in this model predominantly during the early, polymorphonuclear leukocyte-predominant, phase of repair. At this time, the reactive nitrogen intermediates resulting from the metabolism of arginine may mediate some of the events characteristic of early inflammation, including microbiostasis, vasodilation, and inhibition/reversal of platelet aggregation. In turn, the late suppression of this pathway and the catabolism of arginine through arginase may promote macrophage function within wounds.     

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.     

Breeding seasonality among long-tailed macaques,Macaca fascicularis,in Peninsular Malaysia

The females among more than 7000 long-tailed macaques, Macaca fascicularis,that had recently been trapped for biomedical research were examined for pregnancy. The animals were taken from the wild in the north of Peninsular Malaysia during the period 1978 to 1979 and in the south from 1978 to 1982. Both samples showed some pregnancies in all months of the year and a peak during the months of May to July. Additional peaks in other months in some years showed that, while the midyear pregnancy peak was significant, it was not as marked a phenomenon as is the breeding peak in many other species. This might be due to the complexity and unpredictability of both the climate and the macaques’ food supply in Peninsular Malaysia. The additional peaks also illustrated the pitfalls of generalizing about breeding seasonality on the basis of small samples.     

Seasonal occurrence of sea lice Lepeophtheirus salmonis on sea trout in two north Norwegian fjords

Seasonal occurrence of the parasitic copepod Lepeophtheirus salmonis (sea lice) was studied from March to December 2001 in two large north Norwegian sill fjords without fish farming activity, the Ranafjord and the Balsfjord. Anadromous brown trout Salmo trutta (sea trout) in both fjords had a low infestation rate during all sampling periods, but followed a seasonal pattern. During early and late winter (November to December and March to April) and spring (May to June), the prevalence varied from 0 to 25% and the abundance was <0·5 sea lice. Adults dominated (92%) during this period, particularly gravid females. In both fjords, the highest prevalence was during September (80–81%, all stages represented). In Ranafjord, the abundance and mean intensity during this month was 6·8 and 8·6 sea lice, respectively, while in Balsfjord it was 3·6 and 4·5 sea lice, respectively. Fish were captured at temperatures down to 1° C and at full strength sea water which is supposed to cause osmoregulatory problems for the fish. This observation has implications for the understanding of high‐latitude sea trout behaviour and can also make the fish more vulnerable to heavy sea lice infestation during this period. It is suggested that winter running sea trout help to maintain a self replicating local population of sea lice within such fjord systems where other possible hosts ( e.g . farmed Atlantic salmon Salmo salar ) are not present during a whole year cycle.     

Light‐induced pigment degradation in leaves and ripening fruits studied in situ with reflectance spectroscopy

Pigment breakdown mediated by activated oxygen species is a consequence and a general symptom of oxidative stress and injury to plants. We have attempted to estimate the patterns of pigment bleaching and follow pigment susceptibility to irradiation as related to the process of senescence/ripening. Light‐induced pigment breakdown was studied in situ in the leaves of a shade‐requiring plant, wax flower ( Hoya carnosa R. Br.), as well as in apple ( Malus domestica Borlh. cv. Zhigulevskoe) and lemon ( Citrus limon Burm. cv. Pavlovsky) fruits, using reflectance spectroscopy. It was found that the sensitivity of plant pigments to photobleaching increases as ripening progresses in lemon fruit. Kinetic analysis showed that in all systems a rapid breakdown of the pigment occurs after a lag‐phase. The signature analysis revealed a common pattern of chlorophyll and carotenoid changes, but degradation of the individual pigments was found to be inhomogeneous. Both in lemon and apple fruits a decrease in reflectance in the band of carotenoid absorption preceded pigment photodestruction. In the fruits, the bulk of chlorophyll b and the long‐wavelength chlorophyll a forms were degraded at early stages of the process whereas the breakdown of both chlorophylls in H. carnosa leaves was more synchronous. Prolonged irradiation induced bleaching of the main chlorophyll a band with maximum at 678 nm in the difference spectra, as well as carotenoids. Some features of reflectance spectra in the bands of chlorophyll and carotenoid absorption were found to be suitable for the differentiation of photo‐induced pigment breakdown from the transformation of the pigments taking place during senescence.     

Seasonal and daily variations in plasma melatonin in the high-arctic Svalbard ptarmigan (Lagopus mutus hyperboreus).

This study presents the daily rhythm of melatonin secretion throughout one year in a bird from the northern hemisphere, the Svalbard ptarmigan (Lagopus mutus hyperboreus), which lives naturally at 76-80 degrees N. Eight Svalbard ptarmigan were caged outdoors at 70 degrees N and blood sampled throughout one day each month for 13 months. At this latitude, daylight is continuous between May and August, but there is a short period of civil twilight around noon from late November to mid January. There was no daily rhythm in plasma melatonin in May-July. Plasma melatonin levels varied significantly throughout the day in all other months of the year, with the nighttime increase reflecting the duration of darkness. The highest mean plasma concentration occurred at midnight in March (110.1 +/- 16.5 pg/ml) and represented the annual peak in estimated daily production. Around the winter solstice, melatonin levels were significantly reduced at noon and elevated during the nearly 18 h of consecutive darkness, and the estimated mean daily production of melatonin was significantly reduced. Thus, at the times of the year characterized by light-dark cycles, melatonin may convey information concerning the length of the day and, therefore, progression of season. The nearly undetectable low melatonin secretion in summer and the reduced amplitude and production in midwinter indicate a flexible circadian system that may reflect an important adaptation to life in the Arctic.     

Partial purification and characterisation of sucrose synthase in sugarcane

Three sucrose synthase (SuSy) (EC 2.4.1.13) forms were isolated from sugarcane leaf roll tissue. During anion exchange chromatography, one peak of activity (SuSyA) eluted during the wash step and the other peak (SuSyB) during the salt gradient phase at 180mM KCl concentration. A third form of activity (SuSyC), which also eluted at 180mM KCl, was also present in the leaf roll and replaced SuSyB depending on the season of the year. Substrate Km values, as well as sucrose breakdown/synthesis ratios, differed between these forms. For SuSyA, SuSyB, and SuSyC, respectively, Km values+/-SE (mM) were: 41.8+/-3.4, 109+/-23, and 35.9+/-2.3 for sucrose, 1.07+/-0.08, 0.214+/-0.039, and 0.00191+/-0.00019 for UDP, 6.62+/-1.55, 11.7+/-2.6, and 6.49+/-0.61 for fructose, and 3.59+/-0.37, 0.530+/-0.142, and 0.234+/-0.025 for UDP-glucose. Sucrose breakdown/synthesis ratios+/-SE were 0.0791+/-0.0199, 0.330+/-0.180, and 0.426+/-0.069 for SuSyA, SuSyB, and SuSyC, respectively. The ratio of the area of peak 1 (low breakdown/synthesis ratio) to the area of peak 2 (high breakdown/synthesis ratio) in sucrose accumulating tissue (internode 9) was 0.88, while in non-accumulating (leaf roll) tissue it was 14.5 at the same time of year. The molecular mass of the denatured subunits of all three forms was 94kDa by SDS-PAGE. A polyclonal antiserum raised against SuSyB cross-reacted with all three forms on an immunoblot, but only SuSyA and SuSyB were immunoinactivated by this serum.     

Changes in nitrogen levels and free amino acids in rooting cuttings of mulberry (Moms alba)

Total and protein nitrogen in bark and wood of parent stems of mulberry ( Morns alba L. cv. Ichinose) decreased readily and to the same extent during leafing-out of the buds, but the decrease in wood was less marked than in bark. Simultaneously, soluble nitrogen in both bark and wood also declined but the depletion was less marked than that of total and protein nitrogen. During the same period total nitrogen in the new shoots and adventitious roots increased drastically; however, the increase in total nitrogen in the growing parts during rooting was almost the same as the decrease in total nitrogen in the parent stems. Proline, the prevalent amino acid in wood and bark of the parent stems, decreased drastically during rooting, whereas during the same period asparagine in the developing buds, callus and adventitious roots increased markedly and became the predominant amino acid. The amount of arginine was relatively high in bark of the parent stems but Low in wood and the buds. The level of arginine in bark decreased considerably during the experiments (as did that of proline). The results suggest that the nitrogen required by the growing parts (sinks) in the rooting cuttings comes mainly from protein breakdown in bark of the parent stems (source), although stored protein in wood (source) and soluble nitrogen in bark and wood (sources) also play a part in storage of nitrogen. Asparagine is suggested to be the main nitrogen transport compound in the new growth of the tree and the initiating roots of cuttings.     

Reproduction in the round-eared elephant shrew (Macroscelides proboscideus) in the southern Karoo, South Africa

Reproduction in round-eared elephant shrews occurred throughout the year. Male round-eared elephant shrews were spermatogenically active, with spermatozoa stored in the cauda epididymes, throughout the year. Pregnant females occurred in all months except March and May and there was a decline in the occurrence of pregnancies during early winter (March-July). This tendency towards seasonality of pregnancies was mirrored by the appearance of juveniles, with 77% of all young animals caught between September and February. Bone calcium concentrations were significantly lower during the dry winter than during summer. From October onwards (the beginning of the wet season), bone calcium concentrations increased, reaching high levels in December which were maintained until April. This increase in bone calcium concentrations coincided with the rainy season and with an increase in the intake of herbage, and during this period 60% of all pregnancies occurred. We conclude that the ability of the round-eared elephant shrew to vary its diet may allow some individuals to breed throughout the year. The reduction in the occurrence of pregnancies in early winter probably ensures that few births occur when bone calcium concentrations are low.