Evaluation of resectability after neoadjuvant chemotherapy for primary non-resectable colorectal liver metastases: A multicenter study.

BACKGROUND/AIM: The Kyushu Study Group of Clinical Cancer (KSCC) previously reported the safety and efficacy of neoadjuvant chemotherapy with mFOLFOX6 + bevacizumab for H2/H3 liver metastases of colorectal cancer. The aim of the current study was to evaluate the resectability of these metastases before and after chemotherapy as determined by independent liver surgeons. METHODS: Between May 2008 and April 2010, 40 patients were registered in a multicenter phase 2 trial of neoadjuvant chemotherapy (KSCC 0802). In Study 1, 5 independent liver surgeons from five different KSCC centers evaluated the resectability of liver metastases of colorectal cancer based on imaging studies performed before and after chemotherapy. Each surgeon was blinded to the other surgeons' evaluations. In addition, no information about the patients' characteristics was provided. In Study 2, 3 surgeons evaluated the resectability of these lesions based on imaging studies with discussion with each other, with the surgeons being provided with information on the patients' characteristics. RESULTS: In Study 1, 13 patients (36.1%) were evaluated to be resectable at baseline, whereas 17 patients (47.2%) were evaluated to be resectable after chemotherapy. In Study 2, 4 patients (11.1%) were evaluated to be resectable at baseline, compared to 23 patients (63.9%) after chemotherapy. CONCLUSION: Neoadjuvant chemotherapy with mFOLFOX6 + bevacizumab was confirmed to increase the resectability of non-resectable liver metastases of colorectal cancer according to the independent assessments of surgeons.

Carbon-14 transfer into potato plants following a short exposure to an atmospheric 14CO2 emission: observations and model predictions.

To improve the understanding of the environmental (14)C behaviour, the International Atomic Energy Agency (IAEA) coordinated a Tritium and C-14 Working Group (T&C WG) in its EMRAS (Environmental Modelling for Radiation Safety) programme. One of the scenarios developed in the frame of T&C WG involved the prediction of time dependent (14)C concentrations in potato plants. The experimental data used in the scenario were obtained from a study in which potatoes (Solanum tuberosum cv. Romano) were exposed to atmospheric (14)CO(2) in a wind tunnel. The observations were used to test models that predict temporal changes in (14)C concentrations in leaves at each sampling time for each experiment and (14)C concentrations in tubers at the final harvest of each experiment. The experimental data on (14)C dynamics in leaves are poorly reproduced by most of the models, but the predicted concentrations in tubers are in good agreement with the observations.

Rapid uranium preconcentration and separation method from fresh water samples for total U and 235U/238U isotope ratio measurements by ICP-MS.

A simple and rapid method using TRU resin cartridges (Eichrom Technologies, Inc., USA) and quadrupole ICP-MS for total uranium (U) and 235U/238U isotope ratio measurements in fresh water samples was investigated. After U extraction on the resin by sample solution loading, three alkaline reagents, tetramethyl ammonium hydroxide (TMAH), NaOH and NH4OH were studied for U elution behavior from the resin cartridges and applicability of these eluates was evaluated with respect to direct introduction to ICP-MS. Among the studied eluants, TMAH showed the best results with high U recovery and no counting interferences with internal standard elements such as thallium and bismuth. Moreover, U in water samples was separated from many major and minor elements with the TRU cartridges. Almost all U was concentrated in 10 mL of 0.014 M TMAH in 2 h using 200 mL of water sample.

Quistes retro-aeeolares en adolescentes: aspectos clínicos y ultrasonograficos / Retro-areolar cysts in adolescents: clinical and ultrasonographic aspects

Retroareolar cysts are common in pre and postmenarchic girls. Boys are rarely diagnosed with this condition. They correspond to cystic dilatations of the accessory mammary glands that open along with a sebaceous gland at the areola and can be single or multiple, uni or bilateral, palpable or incidental findings on ultrasound. They have variable morphology, thin walls, anechogenic content, sometimes calcic sediment can be observed in their lumen. Infected cysts present enlarged, hypervascularized walls; their content is echogenic, avascular and the adjacent tissue is hyperechogenic, with increased vascularization at color Doppler. If not treated, may become retroareolar abscesses. Inflamatory complications are treated with anti-inflamatory drugs and/or antibiotics. No diagnostic biopsy or puncture aspiration is required, since they are spontaneously drained at the areola. In order to appropriately advise patients and families, it is necessary to have knowledge of both the medical and the ultrasonographic aspects of them and their complications.

Los quistes retroareolares son frecuentes en niñas pre y postmenárquicas. Raramente se diagnostican en el varón. Corresponden a dilataciones quísticas de glándulas mamarias accesorias que se abren junto con una glándula sebácea en la areola, pueden ser únicos o múltiples, uni o bilaterales, palpables o hallazgos incidentales en ecografía. Los no complicados tienen morfología variable, paredes delgadas y contenido anecogénico, pudiendo observarse sedimento calcico en su lumen. Los complicados presentan paredes engrosadas, hipervascularizadas, con contenido ecogénico, avascular, tejidos adyacentes hiperecogénicos y aumento de la vascularización al Doppler color. Sin tratamiento, pueden transformarse en abscesos retroareolares. La complicación inflamatoria se trata con antiinflamatorios y/o antibióticos. No requieren biopsia diagnóstica ni punción evacuadora, puesto que se drenan espontáneamente a la areola. El conocimiento del cuadro clínico y su aspecto ul-trasonográfico permitirá orientar adecuadamente a los pacientes y sus familias.

Use of TEVA resin for the determination of U isotopes in water samples by Q-ICP-MS.

In order to measure uranium isotopic mass ratio in natural water samples by Q-ICP-MS, an application of TEVA resin (Eichrom) was studied to separate and concentrate U. After being evaporated to dryness, the sample residue was dissolved in 6 M HCl, then, TEVA extraction was carried out. U extracted on the resin could be removed with 20 ml of 1 M HCl (U fraction) when Fe content was lower than 2 mg. U recovery in U fraction showed a negative correlation with Fe content in the samples.

Generic values for soil-to-plant transfer factors of radiocesium.

There is a need for soil-to-plant transfer factors of radionuclides that take into account all possible crops on all soil varieties to support dose assessment studies. Because only limited experimental data exist for worldwide soil systems, such values should necessarily have a generic character. This paper describes a generic system for 137Cs, mainly based on a reference soil-to-plant transfer factor which depends solely on soil properties such as nutrient status, exchangeable K-content, pH and moisture content. Crops are divided into crop groups, cereals serving as reference group. The transfer of other crop groups can be calculated by multiplying data for cereals by a conversion factor. Existing data present in the IUR (International Union of Radioecologists) databank and in large part the work of a FAO (Food and Agriculture Organisation)/IAEA(International Atomic Energy Agency)/IUR project on tropical systems provided the basis for the derivation of the conversion factors and reference values.

Effect of acupuncture-like stimulation on cortical cerebral blood flow in anesthetized rats.

The effect of acupuncture-like stimulation of various areas (cheek, forepaw, upper arm, chest, back, lower leg, hindpaw, perineum) on cortical cerebral blood flow (CBF) was examined in anesthetized rats. An acupuncture needle (diameter, 340 microm) was inserted into the skin and underlying muscles at a depth of about 5 mm and twisted to the right and left once a second for 1 min. CBF of the cortex was measured using a laser Doppler flowmeter. Stimulation of the cheek, forepaw, upper arm and hindpaw produced significant increases in CBF, but stimulation of the chest, back, lower leg and perineum did not produce significant responses. Stimulation of the cheek, forepaw, and hindpaw produced an increase in mean arterial pressure (MAP), while stimulation of the back produced a decrease in MAP. Stimulation of the upper arm, chest, lower leg and perineum did not produce a significant MAP response. After spinal transection at the 1st to 2nd thoracic level, the blood pressure response to stimulation of the cheek and forepaw was suppressed, whereas an increase in CBF still took place. The increase in CBF induced by forepaw stimulation was abolished by severance of the somatic nerves at the brachial plexus. Forepaw stimulation enhanced the activity of the radial, ulnar and median nerves. Furthermore, in the present study, passing of an electric current through acupuncture needles showed that excitation of group III (Adelta) and group IV (C) afferent fibers in the somatic nerve was capable of producing an increase in CBF, whereas excitation of group I (Aalpha) and group II (Abeta) fibers was ineffective. The increase in CBF induced by forepaw stimulation was almost abolished by intravenous administration of muscarinic and nicotinic cholinergic blocking agents (atropine 5 mg/kg and mecamylamine 20 mg/kg), and by bilateral lesions in the nucleus basalis of Meynert. Acupuncture-like stimulation of a forepaw increased acetylcholine release in the cerebral cortex. We concluded that the increase in CBF, independent of systemic blood pressure, elicited by acupuncture stimulation is a reflex response in which the afferent nerve pathway is composed of somatic group III and IV afferent nerves, and efferent nerve pathway includes intrinsic cholinergic vasodilators originating in the nucleus basalis of Meynert.

Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Krüppel-like factor, a novel zinc finger repressor.

The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis. Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated KKLF (for "kidney-enriched Krüppel-like factor") and the previously isolated MAZ (for "myc-associated zinc finger protein") were cloned. KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver. In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed. A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG. In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1-luciferase reporter gene. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.

Electro-acupuncture stimulation to a hindpaw and a hind leg produces different reflex responses in sympathoadrenal medullary function in anesthetized rats.

The effects of electro-acupuncture stimulation (EAS) of two different areas of a hindlimb with different stimulus intensities on sympathoadrenal medullary functions were examined in anesthetized artificially ventilated rats. Two needles of 160 microm diameter and about 5 mm apart were inserted about 5 mm deep into a hindpaw (Chungyang, S42) or a hind leg (Tsusanli, S36) and current of various intensities passed to excite various afferent nerve fiber groups at a repetition rate of 20 Hz and pulse duration of 0.5 ms for 30-60 s. Fiber groups of afferent nerves stimulated in a hindlimb were monitored by recording evoked action potentials from the afferents innervating the areas stimulated. The sympathoadrenal medullary functions were monitored by recording adrenal sympathetic efferent nerve activity and secretion rates of catecholamines from the adrenal medulla. EAS of a hindpaw at a stimulus strength sufficient to excite the group III and IV somatic afferent fibers produced reflex increases in both adrenal sympathetic efferent nerve activity and the secretion rate of catecholamines. EAS of a hind leg at a stimulus strength sufficient to excite the group III and IV afferent fibers produced reflex responses of either increases or decreases in sympathoadrenal medullary functions. All responses of adrenal sympathetic efferent nerve activity were lost after cutting the afferent nerves ipsilateral to the stimulated areas, indicating that the responses are the reflexes whose afferents nerve pathway is composed of hindlimb somatic nerves. It is concluded that electro-acupuncture stimulation of a hindpaw causes an excitatory reflex, while that of a hind leg causes either excitatory or inhibitory reflex of sympathoadrenal medullary functions, even if both group III and IV somatic afferent fibers are stimulated.

Isolation and characterization of the human CLC-5 chloride channel gene promoter.

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.

Intermittent administration of human parathyroid Hormone(1-34) prevents immobilization-related bone loss by regulating bone marrow capacity for bone cells in ddY mice.

ddY mice, 6 weeks of age, were neurectomized (Nx) in the right hindlimbs and sham-operated (Sham) in the left limbs for evaluation of the effects of intermittent injections of human parathyroid hormone (hPTH) on trabecular bone turnover and bone marrow cell development in unloaded and loaded limbs. Mice were given subcutaneous injections of hPTH(1-34) five times a week at a dose of 0 (vehicle), 4 (low dose), or 40 (high dose) microg/kg of body weight for 2, 4, or 6 weeks. Histomorphometric analyses of the trabecular bone of the proximal tibiae revealed that high-dose hPTH injections preserved the trabecular bone volume of the Nx limbs, which was reduced after neurectomy, at the same level as that of the contralateral Sham limbs. The mineral apposition rate in the Nx limbs was elevated to values above even that of the Sham limbs by high-dose hPTH injections. The bone formation rate reduced by neurectomy was maintained at the Sham level by low- and high-dose hPTH injections. The neurectomy-induced increase in osteoclast number was suppressed by high-dose hPTH injections. In the bone marrow cells, the numbers of nonadherent and adherent cells per tibia obtained from the Nx and Sham limbs did not change. The hPTH injections decreased the numbers of nonadherent cells and increased those of adherent cells in both the Nx and the Sham limbs, but the effects were less marked in the Nx than in the Sham limbs even at high-dose injections. The formation of osteogenic nodules in the marrow cultures obtained from the Nx limbs was decreased after surgery and was maintained at the level of the Sham limbs by high-dose hPTH injections. The number of osteoclast-like multinucleated cells was reduced in the Sham limbs by high-dose hPTH injections. The value was increased at 2 weeks after neurectomy, but it was maintained at the Sham level by high-dose hPTH injections through the experimental period. The numbers of colony forming units-fibroblastic, which were reduced by neurectomy, and those of colony forming units for granulocytes and macrophages were not altered by hPTH injections. These results demonstrate that intermittent high-dose hPTH administration in the Nx limbs as well as in the contralateral Sham limbs has similar anabolic effects, stimulating osteoblast cell lineage and suppressing osteoclast cell lineage. The anabolic effects at 4 microg were reduced, but the effects at 40 microg seemed to be less affected by unloading due to sciatic neurectomy.

Isolation and characterization of kidney-specific CLC-K2 chloride channel gene promoter.

CLC-K1 and CLC-K2 are highly homologous kidney-specific chloride channels, but they are expressed in the different nephron segments. To understand the molecular mechanisms of kidney-specific and nephron-segment-specific expression of CLC-K channel genes, the rat ClC-K2 gene promoter was cloned and compared with that of CLC-K1. In the 1.5-kb pair 5'-flanking region of the CLC-K2 gene, no TATA box was identified around the transcriptional start site, and the proximal region (-32 to -68) was characterized by a GA-rich motif that had a significant sequence similarity to that of the previously isolated CLC-K1 gene promoter. In contrast, the distal portion did not have significant sequence similarity to that of CLC-K1. Reporter gene assay and gel-retardation analysis revealed that the GA-rich motif and the binding of a specific protein(s) to this element were indispensable for the basal promoter activity of the CLC-K2 gene. These results suggest that the GA-rich element may have an important role in the promoter activities of the kidney-specific CLC-K1 and -K2 genes, but that the GA-element alone is not sufficient for the strict regulation of nephron-segment-specific expression of CLC-K1 and CLC-K2 genes.

Safety and efficacy of autologous platelet transfusion in cardiac surgery: comparison of cryopreservation, blood collection on the day before surgery, and blood collection during surgery.

In a group of 39 patients with ischemic heart and valvular disease (January 1997 to May 1998), three platelet collection methods were compared in terms of safety and effectiveness. The methods were: (i) collection of autologous platelets over several weeks and freezing them for storage until surgery (frozen group, 12 patients); (ii) collection of autologous platelets on the day before surgery and preserving them without freezing (fresh group, 8 patients); and (iii) collection of autologous platelets intraoperatively (intraoperative group, 9 patients). Ten patients served as controls (control group). Blood pressure was not significantly affected by platelet collection in the frozen and fresh groups, but both systolic (P < 0.01) and diastolic blood pressure (P < 0.05) decreased significantly after collecting platelets in the intraoperative group. Similarly, heart rate was unaffected by platelet collection in the frozen and fresh groups, while it increased significantly in the intraoperative group (P < 0.05). Blood loss after 24 h was significantly smaller in the fresh group than in the frozen group (P < 0.05). Total blood transfusion volume was significantly smaller in the frozen and fresh groups than in the intraoperative and control groups (P < 0.05). Bleeding time 2 h postoperatively, when administration of autologous platelets had been completed, was reduced compared with immediately postoperative values in all three groups receiving autologous platelets (P < 0.05). However, only the frozen and fresh groups showed a significantly shorter bleeding time than the control group (P < 0.05). In all three groups receiving autologous platelets, the platelet count was significantly increased after administration of autologous platelets, but only the fresh group had a platelet count that was significantly greater than the control group (P < 0.05). From these results we conclude that the frozen and fresh groups received safer treatment than the intraoperative group. Although hemostasis improved after all three regimes of autologous platelet transfusion, only the frozen and fresh groups had a reduced need for allogeneic blood transfusion compared with the control group. For this reason we conclude that the frozen and fresh groups were also superior to the intraoperative group in terms of effectiveness. However, the recovery of platelets after frozen storage was low, and to obtain a good effect with the freezing method it is necessary to collect and store large volumes of platelets. In terms of simplicity, safety, and efficacy, the fresh method seems to be the preferred technique.

The effect of electro-acupuncture stimulation on the muscle blood flow of the hindlimb in anesthetized rats.

The effect of electro-acupuncture stimulation (EAS) on blood flow in the muscle biceps femoris (MBF) and on mean arterial pressure (MAP) was investigated in anesthetized, artificially ventilated rats. EAS was applied to a hindpaw for 30 s at intensities of 0.1-10.0 mA and at frequencies of 1-20 Hz, and MBF was measured by laser Doppler flowmetry. EAS at less than 1.0 mA, which excited group II fibers maximally and III fibers partially in a saphenous nerve, had no significant effect on MBF or MAP, although both revealed variable responses. EAS at 1.5 mA, which additionally excited group III fibers almost maximally and was subthreshold for group IV fibers, produced a small but significant increase in MBF and MAP. These responses were further increased at 2.0 mA or more, which was suprathreshold for group IV fibers. The increased response of MBF at 10.0 mA was followed by a small decrease in MBF. EAS at 1.5 mA or more also elicited a decrease in renal blood flow (RBF) and an arterial pressor response. Following severance of the bilateral splanchnic nerves, EAS at 10.0 mA induced only a slight increase in MAP and a decrease in MBF. The decrease in MBF was abolished following further severance of the bilateral lumbar sympathetic trunks (LSTs). In conclusion, EAS to a hindpaw at a stimulus strength sufficient to excite group III and IV afferent fibers, particularly group IV afferent fibers, can produce a reflex decrease in MBF via a reflex activation of muscle sympathetic activity, although this decrease in MBF is overridden by an increase in MBF caused passively by a reflex MAP pressor response elicited by a reflex increase, at least in splanchnic sympathetic activity.

Reflex modulation of catecholamine secretion and adrenal sympathetic nerve activity by acupuncture-like stimulation in anesthetized rat.

The effects of acupuncture-like stimulation of the abdomen and a hindlimb on the secretion rates of adrenal medullary catecholamine hormones (adrenaline and noradrenaline) and adrenal sympathetic efferent nerve activity were studied using urethane-anesthetized rats. Acupuncture needles (diameter of 340 microns) were inserted into the skin and underlying muscles of either the abdomen or a hindlimb to a depth of 10 mm and then twisted at a frequency of about 1 Hz for 90 s. The stimuli induced three types of response in both catecholamine secretion and adrenal sympathetic nerve activity (i.e., decrease, increase, and no change). These different responses corresponded with three similar types of response in mean arterial pressure. In spinalized animals, the stimuli produced only increases in both catecholamine secretion and nerve activity, and abdominal stimulation elicited a larger response than hindlimb stimulation. The responses of adrenal nerve activity were eliminated after surgically severing the afferent nerves innervating the abdomen and hindlimb. These findings indicate that the secretion of adrenal medullary hormones is controlled reflexively by acupuncture-like stimulation via excitation of somatic afferent nerves and also via the reflex responses of adrenal sympathetic efferent nerves. Both the spinal cord and supraspinal structures act as reflex centers. The excitatory reflex properties at the propriospinal and segmental levels are modified into a generalized, either excitatory or inhibitory, response by supraspinal structures.

Relationship between molecular weights of pectin and hypocholesterolemic effects in rats.

Hypocholesterolemic activities and other properties of three different molecular weight pectin were examined. The low-molecular-weight pectin (M(r) not equal to 66,000) obtained by decomposition of original pectin (M(r) not equal to 750,000) had the properties of low viscosity and high solubility, but it lost hypocholesterolemic activities in rats. On the other hand, the medium-molecular-weight pectin (M(r) not equal to 185,000) had characteristics of both low viscosity and hypocholesterolemic activities.

Cloning, tissue distribution, and intrarenal localization of ClC chloride channels in human kidney.

Two kidney-specific chloride channels, ClC-K1 and ClC-K2, have been isolated from rat kidney. In the present study, we sought to isolate human homologue of rat ClC-K2 chloride channel that was present in the thick ascending limb of Henle's loop and collecting ducts. Human kidney cDNA library was screened with the whole rat ClC-K2 cDNA probe. Two highly homologous but not identical cDNAs were isolated and sequenced. Northern analysis showed that both clones were expressed only in kidney among various human tissues, demonstrating that kidney-specific ClC family members were also present in human kidney. Because both clones had almost the same nucleotide identity (approximately 80%) with rat ClC-K2, we could not determine by sequence alone which human clone corresponded to rat ClC-K2. Accordingly, we performed reverse transcription PCR using dissected human nephron segments and identified the site of expression of each clone in human nephron segments. One clone was only expressed in the thin limb of Henle's loop and the other was expressed in glomeruli, proximal tubules, and collecting ducts. We identified the latter clone as human ClC-K2 based on the localization of rat ClC-K1 and ClC-K2. Identification of human ClC-K2 clone will be of help in understanding the genetic involvement of chloride channel in disorders of chloride transport such as Bartter's syndrome.

Effects of (-)-epigallocatechin-3-O-gallate (green tea tannin) on the life span of stroke-prone spontaneously hypertensive rats.

1. Effect of (-)-epigallocatechin-3-O-gallate (EGCG), a condensed tannin isolated from green tea leaves, on the life span and hypertensive lesions in the stroke-prone spontaneously hypertensive rat (SHRSP) was compared with that of persimmon tannin. 2. Long-term administration of either 0.5% EGCG or 0.5% persimmon tannin to SHRSP inhibited the incidence of stroke and prolonged the life span, but did not affect the blood pressure. 3. These results indicate that EGCG may prevent incidence of stroke due to the radical scavenging action and inhibition of lipid peroxidation, and may result in prolonging the life span of SHRSP, as in the case of persimmon tannin.

Two isoforms of a chloride channel predominantly expressed in thick ascending limb of Henle's loop and collecting ducts of rat kidney.

Complementary DNAs encoding rat kidney chloride channels (ClC-K2L and ClC-K2S) were isolated by a polymerase chain reaction cloning strategy. Degenerate primers were designed based on the significant amino acid identity of the previously cloned chloride channels (ClC-0, -1, -2, and -K1). The 687-amino acid protein encoded by ClC-K2L is about 80% identical to rat ClC-K1 and about 40% identical to ClC-0, -1, and -2. ClC-K2S encodes a 632-amino acid protein in which 55 amino acids containing the putative second membrane-spanning domain of ClC-K2L are deleted. Chloride currents induced by both clones were very similar in terms of inhibitor sensitivity and anion selectivity (Br- > I- > Cl- >> cyclamate-). Northern blot with total ClC-K2L as a probe under high stringency revealed its message predominantly in kidney, especially in the outer and inner medulla. Reverse transcription polymerase chain reaction technique using microdissected nephron segments revealed that the main site of expression of both clones in kidney was the thick ascending limb of Henle's loop and collecting ducts, where the existence of a variety of chloride channels and their importance for maintaining body fluid homeostasis have been demonstrated. These results suggest that ClC-K2L and -K2S are chloride channels in the thick ascending limb and collecting ducts and may be important routes for transcellular chloride transport like ClC-K1.