Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca(2+) ([Ca(2+)](i)) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca(2+)](i) in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca(2+)](i). Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca(2+)](i). GLP-1 (20 nM) did not affect membrane potential or [Ca(2+)](i). In contrast, acetylcholine (ACh, 100 microM) induced fast membrane depolarization immediately followed by a modest [Ca(2+)](i) increase. When extracellular Ca(2+) was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca(2+)](i) increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP(3)R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca(2+)](i) and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca(2+) through store operated InsP(3)R.     

Dendritic cells from chronic lymphocytic leukemia patients are normal regardless of Ig V gene mutation status

Patients with B-type chronic lymphocytic leukemia (B-CLL) segregate into 2 subgroups based on the mutational status of the immunoglobulin (Ig) V genes and the patients in these subgroups follow very different clinical courses. To examine whether dendritic cells (DCs) generated from CLL patients can be candidates for immune therapy, we compared the phenotypic and functional capacities of DCs generated from patients of the 2 CLL subgroups (normal age-matched subjects [normal-DCs]). Our data show that immature DCs from B-CLL patients (B-CLL-DCs) have the same capacity to take up antigen as those from normal controls. Furthermore, B-CLL-DCs generated from the 2 CLL subgroups up-regulated MHC-II, CD80, CD86, CD83, CD40, and CD54 and down-regulated CD206 in response to stimulation with a cocktail of cytokines (CyC) and secreted increased levels of tumor necrosis factor alpha, interleukin (IL)-8, IL-6, IL-12 (p70), and RANTES in a manner typical of mature normal-DCs. Interestingly, CD54 was significantly more up-regulated by CyC in B-CLL-DCs compared with normal-DCs. Except for CD54, no significant differences in surface molecule expression were observed between normal-DCs and B-CLL-DCs. B-CLL-DCs from both subgroups, including 6 patients with VH1-69, that usually fare poorly, presented tetanus toxoid to autologous T cells in vitro similar to normal- DCs. Our data show that DCs generated from the B-CLL subgroup with unmutated Ig V genes are functionally normal. These results are very promising for the use of DCs from patients with poor prognosis for immunotherapy.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

The relationship between habitat permanence and larval development in California spadefoot toads: field and laboratory comparisons of developmental plasticity

We evaluated differences in larval habitats and life history of three species of spadefoot toads, then compared their life histories in a common garden study. Our field work defined the selective regime encountered by each species. Our Great Basin spadefoot (Spea intermontana) bred asynchronously in permanent streams and springs where there was no risk of larval mortality due to drying. The water chemistry remained fairly stable throughout the larval period. The western spadefoot toad, Sp. hammondii, bred fairly synchronously following heavy spring rains in temporary pools that remained filled an average of 81 d. Fifteen % of the breeding pools dried completely on or before the day the first larvae metamorphosed. The desert spadefoot toad, Scaphiopus couchii, bred synchronously after heavy summer showers in very short duration pools; 62% of the breeding pools dried completely on or before the day the first larvae metamorphosed. The concentration of ammonium nitrogen and CaCO₃ increased markedly as the Sp. hammondii and S. couchii pools dried. S. couchii attained metamorphosis at a much earlier age and smaller size than the other two species. S. couchii also showed little variation in the age at metamorphosis but considerable variation in the size at metamorphosis, while the other two species varied in both age and size. The results identify some variables that could serve as cues of pool drying and demonstrate an association between breeding pool duration, breeding synchrony, development rate, and larval development. Our laboratory study yields information about the genetic basis of the differences in development and controlled comparisons of phenotypic plasticity. We manipulated food supply to study the plastic response of age and size at metamorphosis and hence construct the reaction norm for these variables as a function of growth rate. The growth rates ranged from below to above those observed in natural populations. As in the field, in the lab S. couchii attained metamorphosis at an earlier age and smaller size than the other two species. All three species had a similarly shaped reaction norm for size(y‐axis) and age (x‐axis) at metamorphosis, which was a concave upward curve. A consequence of this shape is that age at metamorphosis changes more readily at low levels of food availability and size at metamorphosis changes more readily at high levels of food availability. If we restrict our observations to just those growth rates that are seen in nature, then S. couchii has almost no variation in the age at metamorphosis but considerable variation in size at metamorphosis, while the other two species vary in both age and size at metamorphosis. All three species increased in size at metamorphosis with increased food levels. Our comparative reaction norm approach thus demonstrates that S. couchii has adapted to ephemeral environments by shifting its growth rate reaction norm so that age at metamorphosis is uniformly fast and is not associated with growth rate. The realized variation is concentrated in size rather than age at metamorphosis.     

The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.     

The effects of acclimation and rates of temperature change on critical thermal limits in Tenebrio molitor (Tenebrionidae) and Cyrtobagous salviniae (Curculionidae)

Critical thermal limits provide an indication of the range of temperatures across which organisms may survive, and the extent of the lability of these limits offers insights into the likely impacts of changing thermal environments on such survival. However, investigations of these limits may be affected by the circumstances under which trials are undertaken. Only a few studies have examined these effects, and typically not for beetles. This group has also not been considered in the context of the time courses of acclimation and its reversal, both of which are important for estimating the responses of species to transient temperature changes. Here we therefore examine the effects of rate of temperature change on critical thermal maxima (CT(max)) and minima (CT(min)), as well as the time course of the acclimation response and its reversal in two beetle species, Tenebrio molitor and Cyrtobagous salviniae. Increasing rates of temperature change had opposite effects on T. molitor and C. salviniae. In T. molitor, faster rates of change reduced both CT(max) (c. 2°C) and CT(min) (c. 3°C), while in C. salviniae faster rates of change increased both CT(max) (c. 6°C) and CT(min) (c. 4°C). CT(max) in T. molitor showed little response to acclimation, while the response to acclimation of CT(min) was most pronounced following exposure to 35°C (from 25°C) and was complete within 24 h. The time course of acclimation of CT(max) in C. salviniae was 2 days when exposed to 36°C (from c. 26°C), while that of CT(min) was less than 3 days when exposed to 18°C. In T. molitor, the time course of reacclimation to 25°C after treatments at 15°C and 35°C at 75% RH was longer than the time course of acclimation, and varied from 3-6 days for CT(max) and 6 days for CT(min). In C. salviniae, little change in CT(max) and CT(min) (<0.5°C) took place in all treatments suggesting that reacclimation may only occur after the 7 day period used in this study. These results indicate that both T. molitor and C. salviniae may be restricted in their ability to respond to transient temperature changes at short-time scales, and instead may have to rely on behavioral adjustments to avoid deleterious effects at high temperatures.     

A new species of Ripipteryx from Belize with a key to the species of the Scrofulosa Group (Orthoptera, Ripipterygidae)

A new species of the genus Ripipteryx (Orthoptera: Tridactyloidea: Ripipterygidae) from the Toledo District of southern Belize is described and illustrated. Ripipteryx mopanasp. n. is placed in the Scrofulosa Group based on its elaborately ornamented frons and is readily distinguished from its congeners by the fusion of the superior and inferior frontal folds to form a nasiform median process, the epiproct with both anterior and posterior margins emarginate, the subgenital plate with distinct lateroapical depressions either side of the median line, the basal plate of the phallus strongly bilobed apically, and the development of well-demarcated denticular lobes in the dorsal endophallic valves. A preliminary key to the species of the Scrofulosa Group is provided.     

Radiological evaluation of bone status in the jaw and in the vertebral column in a group of women

Vertebral bone mineral content was determined in a group of 56 women, ages 30–62. These measurements were compared with the status of supporting bone in the jaws (alveolar, molar and bicuspid) and with gingival health. There was a significant decline in vertebral bone mineral content from the pre- to post-menopausal group. Molar and bicuspid measurements were highly correlated. There was some association between lumbar bone mineral content and molar bone status for postmenopausal women. For postmenopausal women, the cases of greatest percent bone loss in alveolar crest were associated with lower lumbar bone mineral content. Gingival health did not confound the bone status measurements. The 56 subjects did not exhibit the degree of reduction in bone density that is observed in the general population. Further investigation using these radiographic techniques may reveal a link between substantial bone loss in the jaw and moderate to severe bone loss in the lumbar vertebrae.     

Recovery of the Ability to Synthesize DNA in Segments of Normal Size at Long Times After Ultraviolet Irradiation of Human Cells

DNA synthesized in human cells within the first hour after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in nonirradiated cells. The size of these segments approximates the average distance between pyrimidine dimers in the parental DNA. This suggests that the dimers interrupt normal DNA synthesis and result in gaps in the newly synthesized DNA. However, DNA synthesized in human cells at long times after irradiation is made in segments equal or nearly equal to those synthesized by nonirradiated cells. The recovery of the ability to synthesize DNA in segments of normal size occurs in normal human cells, where the dimers are excised, and also in cells of the human mutants xeroderma pigmentosum (XP), where the dimers remain in the DNA. This observation implies that the pyrimidine dimer may not be the lesion that causes DNA to be synthesized in smaller than normal segments.