Genetic analysis of adult leukoencephalopathy patients using a custom-designed gene panel.

Leukoencephalopathies encompass all clinical syndromes that predominantly affect brain white matter. Genetic diagnosis informs clinical management of these patients, but a large part of the genetic contribution to adult leukoencephalopathy remains unresolved. To examine this genetic contribution, we analyzed genomic DNA from 60 Japanese patients with adult leukoencephalopathy of unknown cause by next generation sequencing using a custom-designed gene panel. We selected 55 leukoencephalopathy-related genes for the gene panel. We identified pathogenic mutations in 8 of the 60 adult leukoencephalopathy patients (13.3%): NOTCH3 mutations were detected in 5 patients, and EIF2B2, CSF1R, and POLR3A mutations were found independently in 1 patient each. These results indicate that cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most frequent adult leukoencephalopathy in our cohort. Moreover, brain imaging analysis indicates that CADASIL patients who do not present typical phenotypes may be underdiagnosed if not examined genetically.

Evaluation of a chromogenic agar medium for the detection of extended-spectrum ß-lactamase-producing Enterobacteriaceae.

AIM: To compare the performance of a new chromogenic agar medium CHROMagar ESBL (KC-ESBL) to chromID ESBL (SB-ESBL) for the detection and presumptive identification of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae directly from clinical specimens. METHODS AND RESULTS: A total of 256 specimens were screened for ESBL producers. Also, the genotypes of the ESBLs and plasmid-mediated AmpC ß-lactamases (pAmpCBLs) were characterized by PCR and sequencing. Among the 256 specimens, 17 (6.6%) ESBL producers were isolated on both media. The sensitivity, specificity, positive predictive value and negative predictive value were higher for KC-ESBL (100, 93.3, 51.5 and 100%, respectively) than for SB-ESBL (88.2, 92.9, 46.9 and 99.1%, respectively) (P = 0.72). Enterobacteriaceae harbouring pAmpCBL genes as well as chromosomal cephalosporinase- and penicillinase-hyperproducing Enterobacteriaceae and Pseudomonas aeruginosa accounted for the false-positive results. CONCLUSION: KC-ESBL can detect ESBL producers from clinical specimens with good selectivity and rapid presumptive identification by means of colony colour at 24 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that has evaluated the performance of KC-ESBL that enables the detection and presumptive identification of ESBL producers from clinical specimens.

The Xenopus Sox3 gene expressed in oocytes of early stages.

We have isolated an SRY-type HMG box (Sox) cDNA, XSox3, from a Xenopus immature ovary cDNA library. The XSox3 cDNA contains an open reading frame (ORF) of 309 amino-acid residues, showing 62.7 and 77.0% homology with human and chicken Sox3 proteins, respectively. We also showed that the XSox3 gene is composed of a single exon by sequence analysis of the genomic clone and the determination of the transcription start site of the XSox3. The XSox3 mRNA was detected only in ovary and was at a higher level in immature ovary than in mature ovary. During oocyte maturation, the XSox3 mRNA was most abundant in stage I oocytes, and the XSox3 protein was detected in stage I and II oocytes. Recombinant XSox3 protein produced in Escherichia coli bound specifically to sequences containing the binding motif for the HMG box of SRY or SOX proteins, AACAAT or AACAAAG, demonstrating its sequence-specific DNA binding property. Taken together, these results indicate that the XSox3 protein may participate in early oogenesis of Xenopus as a transcription factor.

A novel Jun N-terminal kinase (JNK)-binding protein that enhances the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1.

We have identified a novel Jun N-terminal kinase (JNK)-binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal-regulated kinase 2 (ERK2) in COS-7 cells. JNKBP1 preferentially interacted with the JNKs, but not with ERK2. Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS-7 cells. JNKBP1 alone had only a marginal effect on JNK activity. However, the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1 was significantly enhanced in the presence of JNKBP1. In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway. These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.

Movement of fluorescein and its glucuronide across retinal pigment epithelium-choroid.

PURPOSE: To characterize movement of fluorescein and its glucuronide across the blood-retinal barrier. METHODS: Retinal pigment epithelium (RPE)-choroid preparations from New Zealand albino rabbit were sealed in an Ussing-type chamber in a stabilized condition for 3 hr, where movement of fluorescein and fluorescein glucuronide across the RPE-choroid was studied under a short circuit condition. RESULTS: The outward (vitreous-choroid) permeability to fluorescein determined at a concentration of 15 mumol/l was about 4 times greater than the inward (choroid-vitreous) permeability (P < 0.01). The outward permeability was significantly decreased by 50-65% by metabolic or competitive inhibitors (1 mumol/l ouabain, 10 mumol/l 2,4-dinitrophenol, 100 mumol/l probenecid, 30 mmol/l hippurate, or 5 mmol/l iodipamide), whereas the inward permeability was not affected by any of the above competitive inhibitors. As the fluorescein concentration was increased from 15 to 150 mumol/l, the net fluorescein movement across the tissue indicated saturation, and a Lineweaver-Burk plot gave an apparent Km of 26 mumol/l and Vmax of 1.56 nmol/hr/cm2. The outward permeability to fluorescein glucuronide determined at 15 mumol/l was about double the inward permeability (P < 0.01) and about 1/3 of the outward permeability to fluorescein (P < 0.01). The outward permeability to fluorescein glucuronide was significantly decreased by about 50% by 1 mumol/l ouabain, 10 mumol/l 2,4-dinitrophenol, or 100 mumol/l probenecid, whereas the inward permeability was not affected by 100 mumol/l probenecid. CONCLUSION: These results suggest that the majority of the outward fluorescein movement across the tissue and part of that of fluorescein glucuronide depends on an active transport mechanism, whereas the inward movement of both fluorescein and fluorescein glucuronide occurs by a passive mechanism.

Neuronal intranuclear inclusions in spinocerebellar ataxia type 2: triple-labeling immunofluorescent study.

Spinocerebellar ataxia type 2 (SCA2) is associated with an expansion of CAG/polyglutamine-repeat of a gene of unknown function. We performed an immunohistochemical study to identify the immunolocalization of the disease protein ataxin-2 in normal and SCA2 patients. Although normal and expanded ataxin-2 were ubiquitously localized to the cytoplasm of neurons, ubiquitinated intranuclear inclusions were observed selectively in 1-2% of neurons of affected brain regions except the cerebellum. Triple-labeling immunofluorescence revealed that ataxin-2, expanded polyglutamine and ubiquitin were colocalized to these neuronal intranuclear inclusions (NIs), indicating that SCA2 shares morphological characteristics common to other neurological disorders associated with an expansion of CAG/polyglutamine-repeat. Lack of NIs in the cerebellar lesion, however, suggests the discrepancy between formation of NIs and neuronal degeneration in SCA2.

Modulation of event-related potentials in normal human subjects by visual divided attention to spatial and color factors.

We investigated how visual event-related potentials (ERPs) are modulated by visual divided attention using an S1-S2 paradigm. Stimulus S2 consisted of non-target stimuli (Stimulus 1, 2, 3) and a target stimulus (Stimulus 4). The spatial/color factor was compared between S1 and S2: same/same (Stimulus 1); same/different (Stimulus 2); different/same (Stimulus 3); and different/different (Stimulus 4). The P1/N1 (90 approximately 150 ms) showed significantly greater amplitude in Stimulus 3 than in Stimuli 1 and 2. The N2 (230 approximately 290ms) showed significantly greater amplitude in Stimulus 2 than in Stimuli 1 and 3. We assumed that the P1/N1 was related to spatial attention, enhanced by alterations to the spatial factor, and that the N2 was related to color attention, enhanced by alterations to the color factor.

Protein-loss into retroperitoneal lymphangioma: demonstration by lymphoscintigraphy and blood-pool scintigraphy with Tc-99m-human serum albumin.

A rare, benign congenital lymphangioma has been reported to occur frequently in the neck and axilla, but rarely in the retroperitoneal space. We report a case of a retroperitoneal lymphangioma associated with hypoproteinemia caused by protein-loss into the tumor. In this case, lymphoscintigraphy with subcutaneously injected Tc-99m-human serum albumin (HSA) disclosed the communication between the tumor and the lymphatic system, and sequential abdominal scintigraphy with intravenously injected Tc-99m-HSA revealed the protein loss into the tumor. Abdominal scintigraphy with Tc-99m-HSA injected intravenously or subcutaneously is occasionally useful for determining the etiology of hypoproteinemia.

Analysis of nucleotide sequence variations in herpes simplex virus types 1 and 2, and varicella-zoster virus.

To analyze the difference in the degree of divergence between genes from identical herpesvirus species, we examined the nucleotide sequence of genes from the herpes simplex virus type 1 (HSV-1) strains VR-3 and 17 encoding thymidine kinase (TK), deoxyribonuclease (DNase), protein kinase (PK; UL13) and virion-associated host shutoff (vhs) protein (UL41). The frequency of nucleotide substitutions per 1 kb in TK gene was 2.5 to 4.3 times higher than those in the other three genes. To prove that the polymorphism of HSV-1 TK gene is common characteristic of herpesvirus TK genes, we compared the diversity of TK genes among eight HSV-1, six herpes simplex virus type 2 (HSV-2) and seven varicella-zoster virus (VZV) strains. The average frequency of nucleotide substitutions per 1 kb in the TK gene of HSV-1 strains was 4-fold higher than that in the TK gene of HSV-2 strains. The VZV TK gene was highly conserved and only two nucleotide changes were evident in VZV strains. However, the rate of nonsynonymous substitutions in total nucleotide substitutions was similar among the TK genes of the three viruses. This result indicated that the mutational rates differed, but there were no significant differences in selective pressure. We conclude that HSV-1 TK gene is highly diverged and analysis of variations in the gene is a useful approach for understanding the molecular evolution of HSV-1 in a short period.

[A rapid determination of allantoin by high-performance liquid chromatography using tris(hydroxymethyl)aminomethane-HCl buffer as a mobile phase].

A rapid and simple method for the determination of allantoin in pharmaceuticals by reversed-phase ion-pair high-performance liquid chromatography using an ODS column was presented. In general, it is difficult to retain allantoin to the ODS column owing to its very low hydrophobicity. We solved these problems by the use of a Tris-HCl buffer (pH 7.5) containing tetra-n-hexyl-ammonium bromide (THAB) as an ion-pair reagent for the mobile phase. Comparatively low concentrations of Tris-HCl buffer (0.9 mM) and THAB (0.5 mM) gave a high capacity factor (k'). As a results of the examination of the chromatographic behavior, it is confirmed that the retention mechanism of allantoin to the ODS column on the present method was not the ion-pair mode, but the ion-exchange mode. Calibration curves for allantoin showed a good linearity in the range of 10 to 400 micrograms/ml (r = 0.9999). The reproducibility (R.S.D., n = 6) was invariably good (0.37%). The lowest concentration of allantoin for the determination was 200 ng per 20 microliters of injection. The present method was successfully applied to the determination of allantoin in commercial eyedrops with good recovery (99.4%). It was found that allantoin in pharmaceuticals could be determined by the present method in short time and without any complicated derivatization.

[Periodic decerebrate spasm with ocular dipping, Cheyne-Stokes respiration and hypersympathetic activity].

Decerebrate spasm is a generalized muscular spasm produced by some stimuli on decerebrate posture. Such spasm are called "tonic fit" or "decerebrate extensor spasm". We reported a 50-year-old man with periodic decerebrate spasm after cerebral hemorrhage. On admission, the patient was comatose. The pupils were round but anisocoric and did not react to light. Corneal reflexes were absent. The face, arms, and legs did not move voluntarily. Two weeks after admission, he was found in decerebrate rigidity. Periodic decerebrate spasms were also observed and were accompanied by ocular dipping. Cheyne-Stokes respiration, and hypersympathetic activity (transiently dilated pupils, hypertension, tachycardia). These symptoms persisted for two months and were induced by painful or sonic stimuli and suppressed by sleep, sedative or antiedematous drugs. The cycle was 0.6 approximately 0.7 per minute in accord with that of Cheyne-Stokes respiration. Magnetic resonance imaging revealed an area of low signal intensity in the midbrain to the bottom of the pons caused by the tentorial herniation on T1-weighted images. From the the clinical features and results of MRI studies, we considered that dysfunction of the midbrain to the pons in addition to diffuse cerebral dysfunction played some role in the manifestation of periodic decerebrate spasm with ocular dipping.

[Disturbance of deep sensation in medial medullary syndrome. Topographical localization of medial lemniscus in the medulla oblongata].

Medial medullary infarction is characterized by ipsilateral hypoglossal nerve palsy with contralateral hemiparesis and disturbance of deep and discriminative sensory perception. We examined the extent and distribution of disturbances in deep sensation and compared the findings with the lesion location in the medial lemniscus detected by MRI in 3 patients with medial medullary infarction. We classified the lesion location into 2 groups; type I and type II. Type I was ventral to the middle medial lesion of the medial lemniscus, and type II was ventral to the dorsal medial lesion. In our series, type I (Case 1) impairment of the three kinds of deep sensations was more severe in the lower extremities than in the up-per extremities. In type II (Cases 2, 3) the severity or impairment in the upper extremities was moderate or severe and nearly equal to that in the lower extremities. There was no difference in the severity of impairment for the four kinds of discriminative sensations. In the literature, type I (8 patients) impairment of position sense in deep sensation was found in 1 of 7 patients in the upper extremities and 5 of 7 patients in the lower extremities. Impairment of vibration sense was found in 1 of 7 patients in the upper extremities and in all patients in the lower extremities. In type II (14 patients) severe impairment of position and vibration sense in deep sensation was found in 3 patients in the upper extremities equal to that in the lower extremities. There was no tendency in the severity of impairment of four kinds of discriminative sensations. Including our 3 cases and 22 in the literature, impairment of deep sensation was more severe in the lower extremities than in the upper extremities in type I (9 patients) and the extent was none (7), mild or moderate (2) in the upper extremities, mild (2), moderate (1), severe (2), obscure (4) in the lower extremities, while in type II (16 patients) the severity in the upper extremities was nearly equal to that in the lower extremities and the extent was none (1), mild or moderate (1), severe (5), obscure (9) in the upper extremities, none (2), mild or moderate (1), severe (6), obscure (7) in the lower extremities. It is concluded that hemiparesis appeared with lesions located in the pyramidal tract of the medulla, hemiparesis and disturbance of deep sensation in the upper and lower extremities, predominantly in the lower extremities with the lesion of the pyramidal tract to the middle of medial lemniscus in the medulla, hemiparesis and disturbance of the upper and lower extremities deep sensation with lesions of the pyramidal tract to the whole of the medial lemniscus in the medulla. Evaluating deep sensation of the upper and lower extremities is useful for speculation of the lesion location in the medial lemniscus in medial medullary infarction.

[A case of Vernet's syndrome due to varicella-zoster virus infection].

We report a 73-year-old man who suffered from an acute onset of dysphagia, cough, hoarseness and left facial and occipital pain. On the 44 days of illness, he was admitted to our clinic. A neurological examination revealed left IX, X and XI cranial nerve palsy. The diagnosis of Vernet's syndrome due to varicella-zoster virus (VZV) infection was made, based on the high titers of VZV antibody in serum. Magnetic resonance imaging revealed a unique nodular lesion with gadolinium enhancement at the medial side of the left jugular foramen. Clinical symptoms improved with intravenous high dose pulse methylprednisolone therapy. The clinical course suggests that the inflammation extended from the left X cranial nerve ganglion.

[Kinetic study of movement of fluorescein across the isolated rabbit retinal pigment epithelium--choroid].

Using an Ussing-type chamber, the transport of fluorescein (F) across the isolated retinal pigment epithelium (RPE)-choroid of the rabbit was studied. The outward movement (from the vitreous to the choroidal side) of F was significantly greater than the inward movement (from the choroidal to the vitreous side) and was suppressed by the application of 10(-4)M probenecid, 30mM hippurate or 5mM iodipamide to 41%, 45% or 39% of the control, respectively, while the inward movement was not affected by any of these agents. As the F concentration in the chamber increased, the inward movement of F also increased in a linear fashion, but the outward movement showed nonlinearity. The difference between the outward and inward movement of F was thought to represent the net flux of F across the RPE-Choroid and this value showed nonlinearity and saturation as the F concentration increased. The Lineweaver-Burk plot of the reciprocals of the net flux of F concentration gave the apparent Km of 4.5 x 10(-5)M and apparent Vmax of 2.27 nmoles/hr/cm2, which suggested that the F transport system in the rabbit RPE-Choroid had a greater affinity to the substrate but lower transporting capacity as compared with the F transport system in the rabbit iris-ciliary body or the ascorbate transport system in the iris-ciliary body.

[Analysis of transport of fluorescein across the isolated retinal pigment epithelium-choroid using an ussing type chamber].

Retinal pigment epithelium (RPE)-choroid preparations from albino rabbits were sealed in an Ussing type chamber under stabilized conditions for 3 hours. The transepithelial potential was 1.2 +/- 0.08 mV and the transepithelial resistance was 175.2 +/- 9.1 omega.cm2 (mean +/- SE, n = 16). The transport of fluorescein across the isolated rabbit RPE-choroid was studied under short circuit condition and outward (vitreous----choroid) and inward (choroid----vitreous) permeability to fluorescein were determined. The outward permeability was 1.63 +/- 0.20 x 10(-5) cm/sec and inward permeability was 0.44 +/- 0.13 x 10(-5) cm/sec (mean +/- SE, n = 8). The former was 4 times greater than the latter (p less than 0.01). The outward permeability was decreased to 1.02 +/- 0.08 x 10(-5) cm/sec (n = 7), 0.75 +/- 0.11 x 10(-5) cm/sec (n = 5), 0.67 +/- 0.11 x 10(-5) cm/sec (n = 6) by 10(-6) M of ouabain, 10(-5) M of 2,4-dinitrophenol and 10(-4) M of probenecid, respectively. Low temperatures (0.5-1.0 degree C) markedly decreased the outward permeability to 0.05 +/- 0.04 x 10(-5) cm/sec (n = 4, mean +/- SE). These results suggest that active transport plays a role in the outward movement of fluorescein across the rabbit RPE-choroid.

[Ocular effects of topical instillation of UF-021 ophthalmic solution in healthy volunteers].

Phase I studies, as divided into two stages, were conducted in healthy volunteers with the ophthalmic solution of UF-021, a novel prostaglandin metabolite-related compound, that was reported to exhibit potent intraocular pressure (IOP)-reducing activity in various species of animals. In the first stage, the vehicle as well as UF-021 ophthalmic solutions at concentration of 0.03%, 0.06% and 0.09% were applied topically to the eyes of 8 healthy volunteers to determine their respective effects through observations on the IOP, and local ocular and systemic side effects. In the second stage, 2 dosages of UF-021 ophthalmic solution, 0.06% and 0.12%, were applied topically to 11 healthy volunteers to investigate the IOP-reducing activities and local ocular side effects. The results revealed that ophthalmic solutions of UF-021 at concentrations ranging from 0.03% to 0.12% reduced IOP in a dose-dependent manner with neither systemic nor local ocular controversial side effects at those dosage levels. In summary, UF-021 ophthalmic solutions, when administered to healthy volunteers through single instillation, reduced IOP significantly without causing any side effects.

[Correlation of clinichopathological features and CAG repeats in SCA2].

Spinocerebellar ataxia type 2 (SCA2) is an inherited neurodegenerative disorder characterized clinically by cerebellar ataxia, slow eye movement, hyporeflexia, involuntary movement, dementia and sensory disturbance and neuropathologically by neuronal loss, mainly in the cerebellar cortex involving all three layers, the pontine nucleus, the inferior olivary nucleus, anterior horn, substantia nigra and thalamus. For making one's diagnosis, it is necessary to give careful consideration to two factors, (age at onset, disease duration). A distinctive neuropathological feature is having both simple atrophy (without degeneration) and numerical atrophy. SCA2 is associated with an expanded CAG repeat that encodes polyglutamine of a gene and a larger number of the repeat is associated with earlier onset and more severe symptoms and more severe neuronal degenerations.

[The effect of long-term physical training on asymptomatic patients with myocardial infarction].

To examine the long-term effect of intense physical training on exercise capacity of patients with myocardial infarction(MI), multiphasic treadmill stress test(MTST) with modified Bruce protocol was performed repeatedly in 10 patients with training(Ex) and in 6 without training(C). In C, heart rate(HR), systolic blood pressure(SBP), or pressure rate product (PRP: HR X SBP/10(-2)) at any stage of MTST was unchanged for 3 years after MI. In Ex, these indices were unchanged at rest, but at stage 4 of MTST decreased significantly as follows: HR 119 +/- 20.4 to 108.6 +/- 16.0/min(p less than 0.05), SBP 151.6 +/- 24.0 to 137.0 +/- 17.5 mmHg(p less than 0.05), PRP 181.9 +/- 49.5 to 153.3 +/- 29.7(p less than 0.05). These indices at maximal exercise increased significantly after one year of training. One year of training in asymptomatic patients with MI reduced of myocardial oxygen consumption index during exercise at given work rate and increased maximal exercise capacity. With continued training these beneficial effects can be maintained for additional 2 years.

Homozygous c.14576G>A variant of RNF213 predicts early-onset and severe form of moyamoya disease.

OBJECTIVE: RNF213 was recently reported as a susceptibility gene for moyamoya disease (MMD). Our aim was to clarify the correlation between the RNF213 genotype and MMD phenotype. METHODS: The entire coding region of the RNF213 gene was sequenced in 204 patients with MMD, and corresponding variants were checked in 62 pairs of parents, 13 mothers and 4 fathers of the patients, and 283 normal controls. Clinical information was collected. Genotype-phenotype correlations were statistically analyzed. RESULTS: The c.14576G>A variant was identified in 95.1% of patients with familial MMD, 79.2% of patients with sporadic MMD, and 1.8% of controls, thus confirming its association with MMD, with an odds ratio of 259 and p < 0.001 for either heterozygotes or homozygotes. Homozygous c.14576G>A was observed in 15 patients but not in the controls and unaffected parents. The incidence rate for homozygotes was calculated to be >78%. Homozygotes had a significantly earlier age at onset compared with heterozygotes or wild types (median age at onset 3, 7, and 8 years, respectively). Of homozygotes, 60% were diagnosed with MMD before age 4, and all had infarctions as the first symptom. Infarctions at initial presentation and involvement of posterior cerebral arteries, both known as poor prognostic factors for MMD, were of significantly higher frequency in homozygotes than in heterozygotes and wild types. Variants other than c.14576G>A were not associated with clinical phenotypes. CONCLUSIONS: The homozygous c.14576G>A variant in RNF213 could be a good DNA biomarker for predicting the severe type of MMD, for which early medical/surgical intervention is recommended, and may provide a better monitoring and prevention strategy.

Movement of carboxyfluorescein across retinal pigment epithelium-choroid.

The movement of carboxyfluorescein (CF) across the isolated retinal pigment epithelium (RPE)-choroid of the albino rabbit was studied using an Ussing chamber under short-circuit conditions with CF concentrations ranging from 15 to 300 microM. The inward (from the choroid to vitreous side) permeability of the tissue to CF showed no significant change over the concentration range tested, averaging 2.2 x 10(-6) cm sec-1. Neither ouabain (1.0 microM), low external sodium concentration nor probenecid (100 microM) had significant effect on the inward movement of CF. The outward (from the vitreous to the choroid side) permeability of the tissue to CF gradually decreased as the concentration increased (ANOVA, P < 0.01: Scheffe's test, P < 0.05), averaging 3.7 x 10(-6) cm sec-1 at 75 microM. Further, it was always significantly greater than the inward permeability at the corresponding concentration (unpaired t-test, P < 0.05-0.01). The outward movement of CF was significantly inhibited by 2,4-dinitrophenol (10 microM), ouabain (1.0 microM), probenecid (100 microM), iodipamide (5.0 mM), hippurate (30 mM) or low external sodium concentration (25 mM). The net outward movement of CF became saturated as concentration was increased and a Lineweaver-Burk plot gave an apparent Km of 237 microM and Vmax of 1.8 nmol hr-1 cm-2. The results indicated that part of the outward movement of CF across the RPE-choroid depends on carrier mediated active transport, but to a much lesser extent than that of fluorescein. The inward movement of CF was thought to occur almost exclusively by passive diffusion through the paracellular spaces.