The boundary of life and death: changes in mitochondrial and cytosolic proteomes associated with programmed cell death of Arabidopsis thaliana suspension culture cells.

Introduction: Despite the critical role of programmed cell death (PCD) in plant development and defense responses, its regulation is not fully understood. It has been proposed that mitochondria may be important in the control of the early stages of plant PCD, but the details of this regulation are currently unknown. Methods: We used Arabidopsis thaliana cell suspension culture, a model system that enables induction and precise monitoring of PCD rates, as well as chemical manipulation of this process to generate a quantitative profile of the alterations in mitochondrial and cytosolic proteomes associated with early stages of plant PCD induced by heat stress. The cells were subjected to PCD-inducing heat levels (10 min, 54°C), with/without the calcium channel inhibitor and PCD blocker LaCl3. The stress treatment was followed by separation of cytosolic and mitochondrial fractions and mass spectrometry-based proteome analysis. Results: Heat stress induced rapid and extensive changes in protein abundance in both fractions, with release of mitochondrial proteins into the cytosol upon PCD induction. In our system, LaCl3 appeared to act downstream of cell death initiation signal, as it did not affect the release of mitochondrial proteins, but instead partially inhibited changes occurring in the cytosolic fraction, including upregulation of proteins with hydrolytic activity. Discussion: We characterized changes in protein abundance and localization associated with the early stages of heat stress-induced PCD. Collectively, the generated data provide new insights into the regulation of cell death and survival decisions in plant cells.

Localization of urea transporter B in the developing bovine rumen.

Urea nitrogen secreted from blood to rumen is a crucial factor shaping the symbiotic relationship between host ruminants and their microbial populations. Passage of urea across rumen epithelia is facilitated by urea transporter B (UT-B), but the long-term regulation of these proteins remains unclear. As ruminal function develops over a period of months, the developing rumen is an excellent model with which to investigate this regulation. Using rumen epithelium samples of calves from birth to 96 d of age, this study performed immunolocalization studies to localize and semi-quantify UT-B protein development. As expected, preliminary experiments confirmed that ruminal monocarboxylate transporter 1 (MCT1) short chain fatty acid transporter protein abundance increased with age (P < 0.01, n = 4). Further investigation revealed that ruminal UT-B was present in the first few weeks of life and initially detected in the basolateral membrane of stratum basale cells. Over the next 2 months, UT-B staining spread to other epithelial layers and semi-quantification indicated that UT-B abundance significantly increased with age (P < 0.01, n = 4 or 6). These changes were in line with the development of rumen function after the advent of solid feed intake and weaning, exhibiting a similar pattern to both MCT1 transporters and papillae growth. This study therefore confirmed age-dependent changes of in situ ruminal UT-B protein, adding to our understanding of the long-term regulation of ruminal urea transporters.

The role of rumen epithelial urea transport proteins in urea nitrogen salvage: A review.

The symbiotic relationship between the host and the rumen microbiome plays a crucial role in ruminant physiology. One of the most important processes enabling this relationship is urea nitrogen salvaging (UNS). This process is important for both maintaining ruminant nitrogen balance and supporting production of their major energy supply, bacterially-derived short chain fatty acids (SCFA). The key step in UNS is the trans-epithelial movement of urea across the ruminal wall and this is a highly regulated process. At the molecular level, the key transport route is via the facilitative urea transporter-B2, localized to ruminal papillae epithelial layers. Additional urea transport through aquaporins (AQP), such as AQP3, is now also viewed as important. Long-term regulation of these ruminal urea transport proteins appears to mainly involve dietary fermentable carbohydrates; whereas, transepithelial urea transport is finely regulated by local conditions, such as CO2 levels, pH and SCFA concentration. Although the key principles of ruminal urea transport physiology are now understood, there remains much that is unknown regarding the regulatory pathways. One reason for this is the limited number of techniques currently used in many studies in the field. Therefore, future research in this area that combines a greater range of techniques could facilitate improvements to livestock efficiency, and potentially, reductions in the levels of waste nitrogen entering the environment.

Inhibition of Urea Transporter (UT)-B Modulates LPS-Induced Inflammatory Responses in BV2 Microglia and N2a Neuroblastoma Cells.

Urea is the major nitrogen-containing product of protein metabolism, and the urea cycle is intrinsically linked to nitric oxide (NO) production via the common substrate L-arginine. Urea accumulates in the brain in neurodegenerative states, including Alzheimer's and Huntington's disease. Urea transporter B (UT-B, SLC14A1) is the primary transport protein for urea in the CNS, identified most abundantly in astrocytes. Moreover, enhanced expression of the Slc14a1 gene has been reported under neurodegenerative conditions. While the role of UT-B in disease pathology remains unclear, UT-B-deficient mice display behavioural impairment coupled with urea accumulation, NO disruption and neuronal loss. Recognising the role of inflammation in neurodegenerative disease pathology, the current short study evaluates the role of UT-B in regulating inflammatory responses. Using the specific inhibitor UTBinh-14, we investigated the impact of UT-B inhibition on LPS-induced changes in BV2 microglia and N2a neuroblastoma cells. We found that UTBinh-14 significantly attenuated LPS-induced production of TNFα and IL-6 from BV2 cells, accompanied by reduced release of NO. While we observed a similar reduction in supernatant concentration of IL-6 from N2a cells, the LPS-stimulated NO release was further augmented by UTBinh-14. These changes were accompanied by a small, but significant downregulation in UT-B expression in both cell types following incubation with LPS, which was not restored by UTBinh-14. Taken together, the current evidence implicates UT-B in regulation of inflammatory responses in microglia and neuronal-like cells. Moreover, our findings offer support for the further investigation of UT-B as a novel therapeutic target for neuroinflammatory conditions.

Evolution of mammalian longevity: age-related increase in autophagy in bats compared to other mammals.

Autophagy maintains cellular homeostasis and its dysfunction has been implicated in aging. Bats are the longest-lived mammals for their size, but the molecular mechanisms underlying their extended healthspan are not well understood. Here, drawing on >8 years of mark-recapture field studies, we report the first longitudinal analysis of autophagy regulation in bats. Mining of published population level aging blood transcriptomes (M. myotis, mouse and human) highlighted a unique increase of autophagy related transcripts with age in bats, but not in other mammals. This bat-specific increase in autophagy transcripts was recapitulated by the western blot determination of the autophagy marker, LC3II/I ratio, in skin primary fibroblasts (Myotis myotis,Pipistrellus kuhlii, mouse), that also showed an increase with age in both bat species. Further phylogenomic selection pressure analyses across eutherian mammals (n=70 taxa; 274 genes) uncovered 10 autophagy-associated genes under selective pressure in bat lineages. These molecular adaptations potentially mediate the exceptional age-related increase of autophagy signalling in bats, which may contribute to their longer healthspans.

Localization of aquaporin-3 proteins in the bovine rumen.

Urea nitrogen salvaging is a crucial mechanism that ruminants have evolved to conserve nitrogen. Facilitative urea transporter-B proteins are known to be involved in urea transport across the rumen epithelium and thus efficiently facilitate the urea nitrogen salvaging process. Recently, functional studies have suggested that aquaglyceroporin transporters might also play a significant role in ruminal urea transport and aquaporin-3 (AQP3) protein has previously been detected in rumen tissue. In this current study, we investigated the specific localization of AQP3 transporters in the bovine rumen. First, end-point reverse-transcription PCR experiments confirmed strong AQP3 expression in both bovine rumen and kidney. Immunoblotting analysis using 2 separate anti-AQP3 antibodies detected AQP3 protein signals at 25, 32, and 42-45 kDa. Further immunolocalization studies showed AQP3 protein located in all the layers of rumen epithelium, especially in the stratum basale, and in the basolateral membranes of kidney collecting duct cells. These data confirm that AQP3 transporters are highly abundant within the bovine rumen and appear to be located throughout the ruminal epithelial layers. The physiological significance of the multiple AQP3 proteins detected and their location is not yet clear, hence further investigation is required to determine their exact contribution to ruminal urea transport.

The power of evolutionary rescue is constrained by genetic load.

The risk of extinction faced by small isolated populations in changing environments can be reduced by rapid adaptation and subsequent growth to larger, less vulnerable sizes. Whether this process, called evolutionary rescue, is able to reduce extinction risk and sustain population growth over multiple generations is largely unknown. To understand the consequences of adaptive evolution as well as maladaptive processes in small isolated populations, we subjected experimental Tribolium castaneum populations founded with 10 or 40 individuals to novel environments, one more favorable, and one resource poor, and either allowed evolution, or constrained it by replacing individuals one-for-one each generation with those from a large population maintained in the natal environment. Replacement individuals spent one generation in the target novel environment before use to standardize effects due to the parental environment. After eight generations we mixed a subset of surviving populations to facilitate admixture, allowing us to estimate drift load by comparing performance of mixed to unmixed groups. Evolving populations had reduced extinction rates, and increased population sizes in the first four to five generations compared to populations where evolution was constrained. Performance of evolving populations subsequently declined. Admixture restored their performance, indicating high drift load that may have overwhelmed the beneficial effects of adaptation in evolving populations. Our results indicate that evolution may quickly reduce extinction risk and increase population sizes, but suggest that relying solely on adaptation from standing genetic variation may not provide long-term benefits to small isolated populations of diploid sexual species, and that active management facilitating gene flow may be necessary for longer term persistence.

UT-B Urea Transporter Localization in the Bovine Gastrointestinal Tract.

Facilitative UT-B urea transporters play an important role in the urea nitrogen salvaging process that occurs in the gastrointestinal tract of mammals, particularly ruminants. Gastrointestinal UT-B transporters have previously been reported in various ruminant species-including cow, sheep and goat. In this present study, UT-B transporter localization was investigated in tissues throughout the bovine gastrointestinal tract. RT-PCR analysis showed that UT-B2 was the predominant UT-B mRNA transcript expressed in dorsal, ventral and cranial ruminal sacs, while alternative UT-B transcripts were present in other gastrointestinal tissues. Immunoblotting analysis detected a strong, glycosylated ~50 kDa UT-B2 protein in all three ruminal sacs. Immunolocalization studies showed that UT-B2 protein was predominantly localized to the plasma membrane of cells in the stratum basale layer of all ruminal sac papillae. In contrast, other UT-B protein staining was detected in the basolateral membranes of the surface epithelial cells lining the abomasum, colon and rectum. Overall, these findings confirm that UT-B2 cellular localization is similar in all ruminal sacs and that other UT-B proteins are located in epithelial cells lining various tissues in the bovine gastrointestinal tract.

Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters.

The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P < 0.01, n = 3, ANOVA) and also increased dimethyl urea-sensitive, arginine vasopressin-stimulated urea flux (P < 0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.01, n = 4, ANOVA) in a concentration-dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P < 0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P < 0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects.

Acute regulation of mUT-A3 urea transporter expressed in a MDCK cell line.

Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) cells. In this study, we engineered a Madin-Darby canine kidney (MDCK) I cell line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A-targeted antibody ML446 detected a approximately 40-kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive (14)C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ cells under basal conditions [not significant (NS), ANOVA]. However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea (P < 0.05, ANOVA) or phloretin (P < 0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intracellular levels of cAMP, via forskolin (P < 0.05, ANOVA), or intracellular calcium, via ATP (P < 0.05, ANOVA). Finally, 1-h preincubation with a specific PKA inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP (P < 0.05, ANOVA). In conclusion, we have produced the first renal cell line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.

Molecular characterization of the mercurial sensitivity of a frog urea transporter (fUT).

The amphibian urea transporter (fUT) shares many properties with the mammalian urea transporters (UT) derived from UT-A and UT-B genes. The transport of urea by fUT is inhibited by the mercurial agent p-chloromercuribenzenesulfonic acid (pCMBS). We found that in oocytes expressing cRNA encoding fUT, a 5-min preincubation in 0.5 mM mercury chloride (HgCl2) also significantly reduced urea uptake. The transport of urea by fUT was rendered mercury (Hg2+) insensitive by mutating either of the residues C185 or H187, both of which lie within the M-I region (close to the hypothetical UT pore). In oocytes expressing a mixture of the C185 and H187 mutants, Hg2+ sensitivity was reestablished. The transport of urea by the mouse UTs mUT-A2 and mUT-A3 was not sensitive to Hg2+. Introducing cysteine residues analogous to that mutated in fUT into mUT-A2 or mUT-A3 did not induce Hg2+ sensitivity. Additionally, introducing the double cysteine, histidine mutations into mUT-A2 or mUT-A3 still did not induce Hg2+ sensitivity, indicating that a region outside of the M-I region also contributes to the Hg2+-induced block of fUT. Using a series of chimeras formed between UT-A3 and fUT, we found that as well as C185 and H187, residues within the COOH terminal of fUT determine Hg2+ sensitivity, and we propose that differences in the folding of this region between fUT and mUT-A2/mUT-A3 allow access of Hg2+ to the fUT channel pore.

Urea nitrogen salvage mechanisms and their relevance to ruminants, non-ruminants and man.

Maintaining a correct balance of N is essential for life. In mammals, the major sources of N in the diet are amino acids and peptides derived from ingested proteins. The immediate endproduct of mammalian protein catabolism is ammonia, which is toxic to cells if allowed to accumulate. Therefore, amino acids are broken down in the liver as part of the ornithine-urea cycle, which results in the formation of urea - a highly soluble, biochemically benign molecule. Mammals cannot break down urea, which is traditionally viewed as a simple waste product passed out in the urine. However, urea from the bloodstream can pass into the gastrointestinal tract, where bacteria expressing urease cleave urea into ammonia and carbon dioxide. The bacteria utilise the ammonia as an N source, producing amino acids and peptides necessary for growth. Interestingly, these microbial products can be reabsorbed back into the host mammalian circulation and used for synthetic processes. This entire process is known as 'urea nitrogen salvaging' (UNS). In this review we present evidence supporting a role for this process in mammals - including ruminants, non-ruminants and man. We also explore the possible mechanisms involved in UNS, including the role of specialised urea transporters.

Characterization of a human colonic cDNA encoding a structurally novel urea transporter, hUT-A6.

Two closely related genes, UT-A (Slc14a2) and UT-B (Slc14a1), encode specialized transporter proteins that modulate the movement of urea across cell membranes. In this article, we report the characterization of a cDNA isolated from human colonic mucosa encoding a novel UT-A urea transporter, hUT-A6. The encoded protein is 235 amino acids (aa) in length, making it the smallest UT-A member characterized. On the basis of previous structural predictions, hUT-A6 is structurally unique in that it consists of a single hydrophobic core flanked by hydrophilic NH(2)- and COOH-terminal domains. The transcript encoding hUT-A6 contains a novel 129-bp exon, exon 5a, which, as a result of alternative splicing, introduces a unique 19-aa segment and a stop codon. Functionally, the protein transports urea, and this activity is inhibited by phloretin. Interestingly, despite the lack of a protein kinase A (PKA) consensus site [RK](2)-X-[ST], transport of urea by hUT-A6 is stimulated by PKA agonists. Deletion of the two PKA consensus sites from murine UT-A3 (mUT-A3) did not affect the stimulatory response of PKA agonists, which, together with the lack of PKA consensus sites in hUT-A6, indicates that regulation of hUT-A6 and mUT-A3 is not mediated through a classic PKA phosphorylation consensus.

Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct.

To investigate the role of inner medullary collecting duct (IMCD) urea transporters in the renal concentrating mechanism, we deleted 3 kb of the UT-A urea transporter gene containing a single 140-bp exon (exon 10). Deletion of this segment selectively disrupted expression of the two known IMCD isoforms of UT-A, namely UT-A1 and UT-A3, producing UT-A1/3(-/-) mice. In isolated perfused IMCDs from UT-A1/3(-/-) mice, there was a complete absence of phloretin-sensitive or vasopressin-stimulated urea transport. On a normal protein intake (20% protein diet), UT-A1/3(-/-) mice had significantly greater fluid consumption and urine flow and a reduced maximal urinary osmolality relative to wild-type controls. These differences in urinary concentrating capacity were nearly eliminated when urea excretion was decreased by dietary protein restriction (4% by weight), consistent with the 1958 Berliner hypothesis stating that the chief role of IMCD urea transport in the concentrating mechanism is the prevention of urea-induced osmotic diuresis. Analysis of inner medullary tissue after water restriction revealed marked depletion of urea in UT-A1/3(-/-) mice, confirming the concept that phloretin-sensitive IMCD urea transporters play a central role in medullary urea accumulation. However, there were no significant differences in mean inner medullary Na(+) or Cl(-) concentrations between UT-A1/3(-/-) mice and wild-type controls, indicating that the processes that concentrate NaCl were intact. Thus, these results do not corroborate the predictions of passive medullary concentrating models stating that NaCl accumulation in the inner medulla depends on rapid vasopressin-regulated urea transport across the IMCD epithelium.

The basolateral expression of mUT-A3 in the mouse kidney.

Facilitative UT-A urea transporters play a central role in the urinary concentrating mechanism. There are three major UT-A isoforms found in the mouse kidney: mUT-A1, mUT-A2, and mUT-A3. The major aim of this study was to identify the location and function of mUT-A3. UT-A proteins were investigated using three novel mouse UT-A-targeted antibodies: ML446, MQ2, and ML194. ML446 detected mUT-A1 and mUT-A3. ML194 detected mUT-A1 and mUT-A2. Importantly, MQ2 was found to be selective for mUT-A3. MQ2 detected a 45- to 65-kDa signal in the mouse kidney inner medulla, which was deglycosylated to a 40-kDa protein band. Immunolocalization studies showed that mUT-A3 was strongly detected in the papillary tip, mainly in the basolateral regions of inner medullary collecting duct (IMCD) cells. Immunoblotting of subcellular fractions of inner medullary protein suggested that in mouse kidney mUT-A3 was present in plasma membranes. Consistent with this, immunoelectron microscopy demonstrated that mUT-A3 was predominantly localized at the basal plasma membrane domains of the IMCD cells in mouse kidney. Heterologous expression of mUT-A3-enhanced green fluorescent protein in Madin-Darby canine kidney cells showed that the protein localized to the basolateral membrane. In conclusion, our study indicates that mUT-A3 is a basolateral membrane transporter expressed in IMCD cells.

Urea movement across mouse colonic plasma membranes is mediated by UT-A urea transporters.

BACKGROUND & AIMS: Urea is a major nitrogen source for commensal bacteria that inhabit the large intestine. UT-A urea transporters mediate urea movement across plasma membranes. The aim of this study was to determine whether UT-A proteins are expressed in the mouse colon and, if so, whether they have a functional role in transcellular urea transport. METHODS: Mouse colonic UT-A transporters were investigated with Northern blot analysis, immunoblotting, immunolocalization, and refractive light flux experiments. RESULTS: Northern blot analysis showed that 4 UT-A transcripts were present in mouse colon. Two peptide-targeted polyclonal antibodies showed the presence of UT-A immunoreactive proteins in mouse colon. Antiserum ML446 targeted to the N-terminus of mouse UT-A1 detected proteins of 34 and 48 kilodaltons. Antiserum ML194 targeted to the C-terminus of mouse UT-A1 detected proteins of 48, 75, and 100 kilodaltons. Immunolocalization studies using ML446 showed the presence of UT-A proteins in cells throughout the colonic crypts. ML194 specifically stained cells located in the proliferative and stem regions of the lower portion of colonic crypts. Differential centrifugation and immunoblotting of colonic epithelia showed that UT-A proteins were present in plasma membrane-enriched fractions. Refractive light flux experiments using colonic plasma membrane vesicles showed a significant urea flux, which was completely inhibited by the UT-A inhibitor phloretin. CONCLUSIONS: Functional UT-A transporters are expressed in the plasma membranes of mouse colon, indicating that these proteins may play a key role in host/bacterial interaction.