Reconstitution of a Regulated Transepithelial Water Pathway in Cells Transfected with AQP2 and an AQP1/AQP2 Hybrid Containing the AQP2-C Terminus

Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9 ± 8.2 vs. 12.5 ± 3.3 cm · sec⁻¹· 10⁻³), but not in cells transfected with AQP1 (15.3 ± 3.6 vs. 13.4 ± 3.6 cm · sec⁻¹· 10⁻³). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly higher than in mock cells at rest (18.1 ± 4.8 vs. 6.7 ± 1.0 cm · sec⁻¹· 10⁻³). The increases in Posm values were not paralleled by increases in ¹⁴C-Mannitol permeability. HgCl₂ inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed, in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder). Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient. These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2. Received: 24 June 1997/Revised: 16 September 1997     

Increased Expression of AQP 1 and AQP 5 in Rat Lungs Ventilated with Low Tidal Volume is Time Dependent

Background and Goals

Mechanical ventilation (MV) can induce or worsen pulmonary oedema. Aquaporins (AQPs) facilitate the selective and rapid bi-directional movement of water. Their role in the development and resolution of pulmonary oedema is controversial. Our objectives are to determine if prolonged MV causes lung oedema and changes in the expression of AQP 1 and AQP 5 in rats.

Methods

25 male Wistar rats were subjected to MV with a tidal volume of 10 ml/kg, during 2 hours (n = 12) and 4 hours (n = 13). Degree of oedema was compared with a group of non-ventilated rats (n = 5). The expression of AQP 1 and AQP 5 were determined by western immunoblotting, measuring the amount of mRNA (previously amplified by RT-PCR) and immunohistochemical staining of AQPs 1 and 5 in lung samples from all groups.

Results

Lung oedema and alveolar-capillary membrane permeability did not change during MV. AQP-5 steady state levels in the western blot were increased (p<0.01) at 2 h and 4 h of MV. But in AQP-1 expression these differences were not found. However, the amount of mRNA for AQP-1 was increased at 2 h and 4 h of MV; and for AQP 5 at 4 h of MV. These findings were corroborated by representative immunohistochemical lung samples.

Conclusion

In lungs from rats ventilated with a low tidal volume the expression of AQP 5 increases gradually with MV duration, but does not cause pulmonary oedema or changes in lung permeability. AQPs may have a protective effect against the oedema induced by MV.     

Effect of month and season of calving on the length of subsequent calving interval in Nili-Ravi buffaloes

Data pertaining to records of 1,274 calving intervals for 400 breeding buffaloes maintained at the Livestock Experiment Station, Qadirabad, District Sahiwal (Pakistan), were analyzed to investigate the magnitude of influence of month and season of calving on the length of subsequent calving interval. The longest average calving interval of 609.9 days was recorded for buffaloes calving in January, while the shortest average calving interval of 502.8 days was observed for those calving in June, the difference being highly significant (P < 0.01). When the data were split in accordance with season of calving, the calving intervals for buffaloes calving in winter, spring, summer and autumn averaged 569.1, 570.6, 506.6 and 515.7 days, respectively. The differences between intervals for winter and spring calvings and between intervals for summer and autumn calvings were non-significant. All other differences were highly significant (P < 0.01). Thus, in most of the buffaloes calving in winter or spring, the first post-partum oestrus and conception are delayed till the following autumn or winter, resulting in significantly longer calving intervals. In contrast, animals calving in summer or autumn exhibit post-partum heat and conception during autumn or winter, resulting in shorter calving intervals.     

Seasonal fluctuations in the response of Italian Mediterranean buffaloes to synchronization of ovulation and timed artificial insemination

A comprehensive study of the efficiency of synchronization of ovulation and timed artificial insemination (TAI) was undertaken in a large group of Italian Mediterranean buffaloes at a commercial dairy. A total of 2791 synchronization protocols were carried out on 857 animals over 3 years. Of these protocols, 823 (29.5%) did not proceed beyond Day 7 (due to the absence of a vascularized CL) and 620 (22.2%) were discontinued on Day 10 (due to the absence of follicles >1.0 cm and tonic uteri); hence, 1443 (51.7%) protocols did not progress to TAI. Data were analyzed for four periods: P1, transition to spring (from breeding season to low breeding season); P2, low breeding season; P3, transition to fall (low breeding season to breeding season); and P4, breeding season. No differences were found among the four periods in terms of the proportion of protocols that did not result in TAI. Of the 857 buffaloes, 660 (77%) conceived and delivered a calf. The average number of TAI per pregnancy was 2.1 and ranged from 1.9 to 2.3 across years. Logistic regression analysis showed that buffaloes that calved during P3 had a higher odds ratio for pregnancy (1.380; P < 0.05) than buffaloes that calved in other periods. Pregnancy was also influenced by the calving to service period (odds ratio = 0.977; P < 0.01) and the pregnancy per AI (P/AI) at successive TAI (odds ratio = 1.480; P < 0.01). The pregnancy per AI at the first TAI (424/857, 49.5%) was greater (P < 0.01) than in subsequent TAI. The occurrence of late embryonic mortality (between Days 27 and 45 after TAI) was similar among the four periods. These findings indicated that there are distinct seasonal differences in the response of Italian Mediterranean buffaloes to synchronization and TAI.     

Age-related and seasonal variations in plasma uncarboxylated osteocalcin in male Murrah buffaloes

The present study aims to investigate the age-related and seasonal variations in plasma uncarboxylated osteocalcin (UcOCN) in male Murrah buffaloes. In experiment 1, body weight, body dimensions and plasma UcOCN levels were estimated in 120 male Murrah buffaloes aged between 18 days and 8 years. We observed a significant (p < 0.0001) decrease in plasma UcOCN with age and their levels ranged between 0.25 and 9.25 ng/ml. In experiment 2, the plasma UcOCN, cortisol, triiodothyronine (T₃), thyroxine (T₄) and prolactin levels were determined during spring and summer in 6–34 months (n = 24) old male Murrah buffaloes. Significant (p < 0.0001) higher plasma UcOCN levels were observed in spring than in summer. The plasma T₃ and T₄ levels were positively correlated with plasma UcOCN, whereas the plasma cortisol and prolactin were negatively correlated. Further, to observe the effect of season on temporal variations of UcOCN, Murrah buffaloes aged between 4–6 (n = 5), 14–16 (n = 5) and 28–30 (n = 5) months were bled at an interval of 30 min, between 0700 and 01030 h, during both the seasons. We observed significant effects of season (p < 0.0001), age (p < 0.0001) and season-by-age interaction (p < 0.01) on plasma UcOCN. The reasons for variations in plasma UcOCN with age and season will be discussed.     

Identification of a keratinocarcinoma cell line expressing AQP3

BACKGROUND INFORMATION: AQP3 (aquaporin 3) in the skin is important for skin moisture as demonstrated by the studies of AQP3-null mice, which have accelerated skin drying. Prevention of dry skin is important not only from a cosmetic but also from a clinical point of view. Primary keratinocyte cultures are cumbersome for screening substances that modulate AQP3 expression. RESULTS: A human keratocarcinoma cell line was found to express AQP3 mRNA and protein, which responded to hypertonic stimulation with sorbitol, suggesting that the AQP3 expression is normally regulated in this cell line. This cell line also expressed the type 1 keratinocyte transglutaminase gene. The AQP3 expression was unaffected by all-trans-retinoic acid up to 10(-6) M. Similarly, the retinoic acid did not increase the AQP3 expression up to 1% concentration in rat skin. CONCLUSION: This cell line is useful for the screening of candidate substances that modulate AQP3 expression.     

Water channel protein AQP3 is present in epithelia exposed to the environment of possible water loss.

Aquaporins (AQPs) are membrane water channel proteins expressed in various tissues in the body. We surveyed the immunolocalization of AQP3, an isoform of the AQP family, in rat epithelial tissues. AQP3 was localized to many epithelial cells in the urinary, digestive, and respiratory tracts and in the skin. In the urinary tract, AQP3 was present at transitional epithelia. In the digestive tract, abundant AQP3 was found in the stratified epithelia in the upper part, from the oral cavity to the forestomach, and in the simple and stratified epithelia in the lower part, from the distal colon to the anal canal. In the respiratory tract, AQP3 was present in the pseudostratified ciliated epithelia from the nasal cavity to the intrapulmonary bronchi. In the skin, AQP3 was present in the epidermis. Interestingly, AQP3 was present at the basal aspects of the epithelia: in the basolateral membranes in the simple epithelia and in the multilayered epithelia at plasma membranes of the basal to intermediate cells. During development of the skin, AQP3 expression commenced late in fetal life. Because these AQP3-positive epithelia have a common feature, i.e., they are exposed to an environment of possible water loss, we propose that AQP3 could serve as a water channel to provide these epithelial cells with water from the subepithelial side to protect them against dehydration. (J Histochem Cytochem 47:1275-1286, 1999)     

Physiological roles of AQP7 in the kidney: Lessons from AQP7 knockout mice

The aquaporin7 (AQP7) water channel is known to be a member of the aquaglyceroporins, which allow the rapid transport of glycerol and water. AQP7 is abundantly present at the apical membrane of the proximal straight tubules in the kidney. In this paper, we review the physiological functions of AQP7 in the kidney. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP7 knockout mice compared to wild-type mice (AQP7, 18.0 ± 0.4 × 10⁻³ cm/s vs. wild-type, 20.0 ± 0.3 × 10⁻³ cm/s). Although AQP7 solo knockout mice did not show a urinary concentrating defect, AQP1/AQP7 double knockout mice showed reduced urinary concentrating ability compared to AQP1 solo knockout mice, indicating that the contribution of AQP7 to water reabsorption in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glycerol in their urine (AQP7, 1.7 ± 0.34 mg/ml vs. wild-type, 0.005 ± 0.002 mg/ml). This finding identified a novel pathway of glycerol reabsorption that occurs in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic-reperfusion ARF model, an increase of urine glycerol was observed (pre-treatment, 0.007 ± 0.005 mg/ml; cisplatin, 0.063 ± 0.043 mg/ml; ischemia, 0.076 ± 0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.     

Seasonal shifts in the thermoregulatory behaviour of australian blacksnakes, Pseudechis porphyriacus (serpentes: elapidae)

1. 1.Activity patterns, movements and thermoregulation of 27 adult blacksnakes containing miniature temperature-sensitive radiotransmitters were monitored in spring and summer in a montane area of eastern Australia.

2. 2.Within each season, mean selected body temperatures were similar in the two sexes, regardless of reproductive status.

3. 3.In spring, male temperatures tended to be more variable than those of females, because the long-range movements of males precluded precise thermoregulation.

4. 4.Although ambient (air) temperatures were higher in summer than in spring, the reverse was true of body temperatures: both male and female snakes were approx. 5°C cooler in summer than in spring.

5. 5.Snakes were less active in summer than in spring, and were cooler in summer even when they were active.

6. 6.We attribute the decreased activity levels in summer to drought, which apparently depressed availability of prey (frogs), and hence foraging by the snakes. Under such circumstances, the metabolic costs of maintaining high body temperatures may exceed the likely benefits of unproductive foraging.

Dynamic and energetic mechanisms for the distinct permeation rate in AQP1 and AQP0

Despite sharing overall sequence and structural similarities, water channel aquaporin 0 (AQP0) transports water more slowly than other aquaporins. Using molecular dynamics simulations of AQP0 and AQP1, we find that there is a sudden decrease in the distribution profile of water density along the pore of AQP0 in the region of residue Tyr23, which significantly disrupts the single file water chain by forming hydrogen bond with permeating water molecules. Comparisons of free-energy and interaction-energy profiles for water conduction between AQP0 and AQP1 indicate that this interruption of the water chain causes a huge energy barrier opposing water translocation through AQP0. We further show that a mutation of Tyr23 to phenylalanine leads to a 2- to 4-fold enhancement in water permeability of AQP0, from (0.5 ± 0.2) × 10^− 14 cm³s^− 1 to (1.9 ± 0.6) × 10^− 14 cm³s^− 1. Therefore, Tyr23 is a dominate factor leading to the low water permeability in AQP0.     

Localization and expression of AQP5 in cornea, serous salivary glands, and pulmonary epithelial cells

Aquaporin (AQP) 5 gene was recently isolated from salivary glandand identified as a member of the AQP family. The mRNAexpression and localization have been examined in several organs. Thepresent study was focused on elucidation of AQP5 expression andlocalization in the eye, salivary gland, and lung in rat. RNaseprotection assay confirmed intense expression of AQP5 mRNA in theseorgans but negligible expression in other organs. To examine the mRNA expression sites in the eye, several portions were microdissected fortotal RNA isolation. AQP5 mRNA was enriched in cornea but not in otherportions (retina, lens, iris/ciliary body, conjunctiva, or sclera).AQP5 was selectively localized on the surface of corneal epithelium inthe eye by immunohistochemistry and immunoelectron microscopy using anaffinity-purified anti-AQP5 antibody. AQP5 was also localized on apicalmembranes of acinar cells in the lacrimal gland and on the microvilliprotruding into intracellular secretory canaliculi of the seroussalivary gland. In the lung, apical membranes of type I pulmonaryepithelial cells were also immunostained with the antibody. Thesefindings suggest a role of AQP5 in water transport to preventdehydration or to secrete watery products in these tissues.

     

牦牛水通道蛋白AQP1和AQP3基因克隆及在不同组织中表达和定位

水通道蛋白（Aquaporin,AQP）广泛存在于生物体的各组织部位,影响着生物体水代谢的过程。为进一步研究水通道蛋白1（AQP1）和水通道蛋白3（AQP3）生物学功能,本文对牦牛（Bos grunniens）不同组织中AQP1和AQP3基因的表达与定位进行了研究。采用PCR方法扩增牦牛AQP1和AQP3基因,对其序列进行生物信息学分析,采用qPCR方法检测2个基因在牦牛不同种组织中的表达量,采用免疫组织化学的方法分析AQP1和AQP3蛋白在组织中的定位和表达。结果表明,牦牛AQP1和AQP3基因CDS区分别为816 bp和840 bp,与野牦牛的同源性最高为99%。AQP1和AQP3基因的表达量在肾脏中最高,显著高于心脏、肝脏、脾脏、肺脏、肌肉、小肠和瘤胃,AQP1基因在各组织中的表达均高于AQP3基因的表达。AQP1和AQP3蛋白定位发现其主要分布于肾脏近曲小管、小肠固有层细胞和瘤胃颗粒层细胞中,均在肾脏中的表达量最高。AQP1蛋白在脾脏、小肠和肌肉组织中的表达极显著高于AQP3蛋白表达。该研究结果对高寒低氧环境中动物的研究具有借鉴意义,有助于牦牛AQP1和AQP3功能研究。     

Expression of HSP70 genes in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle during different seasons under tropical climatic conditions

Skin is most important environmental interface providing a protective envelope to animals. It's always under the influence of both internal and external stressors. Heat shock proteins (HSP) are highly conserved stress proteins which play crucial roles in environmental stress tolerance and thermal adaptation. Present study was planned to observe the relative mRNA expression of inducible (HSP70.1 and HSP70.2) and constitutive (HSP70.8) HSP in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle during different seasons. Skin biopsies were collected from rump region of each animal, aseptically during winter, spring and summer season. Quantitative real time polymerase chain reaction was performed to examine the gene expression of constitutive (HSP70.8) and inducible (HSP70.1 and HSP70.2) HSP in skin of both the breeds during different seasons. Present study observed higher expression of both constitutive and inducible HSP genes in both the breeds during summer and winter than spring season, but magnitude of increase was higher during summer than winter. During summer season, expression pattern of HSPs in skin showed breed differences, where constitutive HSP expression was higher in Tharparkar than Karan Fries and that of inducible HSP was higher in Karan Fries than Tharparkar. Hence, present study suggested that HSP may be conveniently used as biomarkers for assessing protective response of skin against heat stress in zebu and crossbred cattle. Variation in expression between breeds is associated with their heat tolerance and thermal adaptability. In summary, skin of zebu cattle (Tharparkar) is more resistant to summer stress than crossbred (Karan Fries), providing greater protection against heat stress during summer season. Superior skin protective mechanism of zebu (Tharparkar) than crossbred (Karan-Fries) cattle against heat stress may contribute to superior adaptability of zebu cattle to tropical climatic conditions than crossbreed.     

Prolonged Ethanol Ingestion Increases Renal AQP2 and AQP3 Expression in Adult Ratsand in Their Offspring

This study evaluates the effect of prolonged ethanol ingestion on the renal ability to concentrate urine. Suckling Wistar rats born to mothers given ethanol before and during gestation and suckling periods (ethanol-exposed offspring) were used and the results were compared with those obtained from offspring of dams given diets containing no ethanol. Comparisons were also made between progenitors with or without prolonged ethanol ingestion. Body and kidney weights; arginine-vasopressin (AVP) and aldosterone plasma levels; plasma, urine and renal papillary osmolality; urine outflow; kidney AQP2, AQP3 and AQP4 expression and diencephalon AVP mRNA expression were determined. As compared with control offspring, the ethanol-exposed offspring present i) lower body and kidney weights; ii) lower urine outflow; iii) higher renal AQP2 and AQP3 mRNA; iv) higher renal AQP2 protein content and v) higher urine and renal papillary osmolality. These changes were also observed in the ethanol-treated progenitors, although they were of smaller magnitude. Plasma osmolality, renal AQP4 mRNA, AVP plasma levels and diencephalon AVP mRNA expression were not affected by the ethanol treatment. Plasma levels of aldosterone were only significantly increased in the ethanol-exposed suckling rats. It is concluded that maternal ethanol ingestion before and during gestation and suckling periods affects the renal function of the offspring, up-regulating renal AQP2 expression by an AVP-independent mechanism. Ethanol-treated progenitors manifest similar renal changes, although of lesser magnitude than the offspring.     

Osmotic concentration in three races of honey bee,Apis mellifera L. under environmental conditions of arid zone

Hemolymph osmolarity has great effect on honey bee health, especially in arid and semi-arid zones. It regulates water and nutrients in stressed tissues. Osmotic concentration in three races (Apis mellifera ligustica, A. m. carnica and A. m. jemenitica) of Apis mellifera was tested in central Saudi Arabia during spring and summer seasons in 2015. Newly emerged bee workers were first marked and later their hemolymph was extracted after intervals of 1, 5, 10, 15, 20 and 25 days. A significant positive correlation between age and osmolarity was found in all three races during spring and summer seasons. The lowest combined osmotic concentration for all three races was found after 1 day interval, while the highest osmotic concentration was recorded after 25 days. Among all races, A. m. ligustica showed significantly high osmotic concentration after 25 days in spring and summer seasons as compared to the other two races. Only A. m. jemenitica showed similar osmotic concentration after 10 and 15 days in both spring and summer seasons compared to other two races. Mean osmotic concentration of all three races was significantly different after 20 and 25 days in spring and summer seasons. Overall mean recorded during summer was significantly higher than the mean of spring season. Combined osmotic concentration in young drones of all races was significantly lower than that of old drones during spring and summer seasons.     

Spatiotemporal pattern of local sexual segregation in a tree-dwelling temperate bat Myotis daubentonii

Sexual segregation is a common phenomenon in temperate bats, but little is known about driving forces or spatiotemporal patterns. Sexual segregation of the tree-dwelling Daubenton’s bat (Myotis daubentonii) was analyzed. Day roosts and foraging areas of radio-tracked animals were recorded during late spring, early summer, and late summer. Home ranges were calculated for each sex based on identified locations. Distance analysis between sex-specific roosts and foraging areas was conducted for each period. Mixed colonies were confirmed by trapping at roosts. Home range of males was larger (47.3 km²) than of females (9.7 km²). During late spring, distances between male and female roosts were larger (median = 715 m) than during early (median = 474 m) and late summer (median = 489 m). Distances between sex-specific foraging areas were larger during early summer (median = 2,134 m) than during both late spring (median = 1,116 m) and late summer (median = 628 m). The proportion of males in mixed groups increased from late spring (0.2 ± 0.1) to late summer (0.5 ± 0.2). These results show that sexual segregation is not static, but depends on energetic demand of individuals and mating willingness. A high energetic demand in females and low mating willingness in both sexes leads to distinct sexual segregation during late spring. When mating willingness increases, despite an increasing energy demand in males, sexes aggregate to mate.     

Identifying activity patterns from activity counters in ETHOSYS® collars on red deer

Identifying activity patterns of animals from activity counts recorded by sensors in the storage-telemetry-system ETHOSYS^® requires to calibrate activity counters to field activities. We continuously observed from dawn to dusk during days 1, 3 and 5 three collared red deer hinds introduced into a 1-ha enclosure for five consecutive days in February (winter), May (spring) and July (summer) 2001. On the basis of concurrently count data collected every 5 min for each hind and each season, we selected the threshold sensor values which best allowed separation of inactive (mainly in the lying position) and active bouts. In winter and spring, the threshold sensor values ranged from 5 to 20 counts per 5 min, which allowed to correctly classified 90.1–98.5% of diurnal sampling intervals. However, higher threshold values (from 55 to 110 counts per 5 min) were found in summer, because foraging and lying periods occur more frequently together within the 5-min interval counts. So, only 76.9–81.4% of sampling intervals were correctly discriminated. To compare the daily activity patterns of hinds in each season, the percentage of time spent active was calculated on a hourly basis. Synchronized periods of activity occur among the three hinds throughout a 24-h period both in winter and summer. However, especially during daytime, the activity patterns of hinds differed significantly in spring. This could affect interpretation of activity patterns of animals from activity counts averaged from several individuals, even if having similar status and environmental conditions or over periods longer than the interval count adopted in the present study.