Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium.

White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Biochemical characterization of hydroquinone hydroxylase from Phanerochaete chrysosporium.

The white-rot fungus Phanerochaete chrysosporium can degrade lignin polymers using extracellular, non-specific, one-electron oxidizing enzymes. This results in the formation of guaiacyl (G), syringyl (S), and hydroxyphenyl (H) units, such as vanillic acid, syringic acid, and p-hydroxybenzoic acid (p-HBA) and the corresponding aldehydes, which are further metabolized intracellularly. Therefore, the aim of this study was to identify proteins involved in the hydroxylation of H-unit fragments such as p-HBA and its decarboxylated product hydroquinone (HQ) in P. chrysosporium. A flavoprotein monooxygenase (FPMO), PcFPMO2, was identified and its activity was characterized. Recombinant PcFPMO2 with an N-terminal polyhistidine tag was produced in Escherichia coli and purified. In the presence of NADPH, PcFPMO2 used six phenolic compounds as substrates. PcFPMO2 catalyzed the hydroxylation of the H-unit fragments such as p-HBA and HQ, and the G-unit derivative methoxyhydroquinone (MHQ). The highest catalytic efficiency (kcat/Km) was observed with HQ, indicating that PcFPMO2 could be involved in HQ hydroxylation in vivo. Additionally, PcFPMO2 converted MHQ to 3-, 5-, and 6-methoxy-1,2,4-trihydroxybenzene (3-, 5-, and 6-MTHB), respectively, suggesting that PcFPMO2 might partially be involved in MHQ degradation, following aromatic ring fission, via three MTHBs. FPMOs are divided into eight groups (groups A to H). This is the first study to show MHQ hydroxylase activity of a FPMO-group A superfamily member. These findings highlight the unique substrate spectrum of PcFPMO2, making it an attractive candidate for biotechnological applications.

Biochemical Characterization of a Pectate Lyase AnPL9 from Aspergillus nidulans.

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization > 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0-11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family.

Characterization of two 1,2,4-trihydroxybenzene 1,2-dioxygenases from Phanerochaete chrysosporium.

Lignin is the most abundant aromatic compound in nature, and it plays an important role in the carbon cycle. White-rot fungi are microbes that are capable of efficiently degrading lignin. Enzymes from these fungi possess exceptional oxidative potential and have gained increasing importance for improving bioprocesses, such as the degradation of organic pollutants. The aim of this study was to identify the enzymes involved in the ring cleavage of the lignin-derived aromatic 1,2,4-trihydroxybenzene (THB) in Phanerochaete chrysosporium, a lignin-degrading basidiomycete. Two intradiol dioxygenases (IDDs), PcIDD1 and PcIDD2, were identified and produced as recombinant proteins in Escherichia coli. In the presence of O2, PcIDD1 and PcIDD2 acted on eight and two THB derivatives, respectively, as substrates. PcIDD1 and PcIDD2 catalyze the ring cleavage of lignin-derived fragments, such as 6-methoxy-1,2,4-trihydroxybenzene (6-MeOTHB) and 3-methoxy-1,2-catechol. The current study also revealed that syringic acid (SA) was converted to 5-hydroxyvanillic acid, 2,6-dimethoxyhydroquinone, and 6-MeOTHB by fungal cells, suggesting that PcIDD1 and PcIDD2 may be involved in aromatic ring fission of 6-MeOTHB for SA degradation. This is the first study to show 6-MeOTHB dioxygenase activity of an IDD superfamily member. These findings highlight the unique and broad substrate spectra of PcIDDs, rendering it an attractive candidate for biotechnological application. KEY POINTS: • Novel intradiol dioxygenases (IDD) in lignin degradation were characterized. • PcIDDs acted on lignin-derived fragments and catechol derivatives. • Dioxygenase activity on 6-MeOTHB was identified in IDD superfamily enzymes.

Intracorporeal hemi-hand-sewn technique for end-to-end anastomosis in laparoscopic left-side colectomy.

BACKGROUND: Recently, complete laparoscopic procedures with intracorporeal reconstruction were performed in laparoscopic colectomies; however, they were scarcely reported in left-side colectomies because of the anatomical reasons. Since the descending colon is extensively fixed to the retroperitoneum, the dissection range required for resection cannot always be enough for a safe extracorporeal anastomosis. We devised an intracorporeal hemi-hand-sewn (IC-HHS) technique for end-to-end anastomosis in laparoscopic left-side colectomies. MATERIALS AND METHODS: A total of 11 patients underwent IC-HHS anastomosis for the treatment of colon cancer around the sigmoid-descending (SD) junction. The posterior wall of the anastomosis was constructed with a linear stapler and subsequently, the anterior wall was sutured with an intracorporeal hand-sewn technique. Perioperative outcomes were evaluated. RESULTS: IC-HHS reconstruction between the descending colon and sigmoid colon was performed in 11 cases. There were six males and five females with an average age of 66.5 years. The average body mass index was 26.1 kg/m2. The averages of the operation time and intraoperative blood loss were 181.2 min (range, 154 to 210 min) and 13.9 ml (range 5-30 ml), respectively. There were no perioperative complications except for one patient with a superficial surgical site infection. CONCLUSIONS: IC-HHS anastomosis was successfully performed for colon cancer around the SD junction with acceptable perioperative outcomes and there were no procedure-related complications, indicating its feasibility. IC-HHS anastomosis could eliminate unnecessary splenic flexure mobilization in left-side colectomies. IC-HHS anastomosis can be an optional reconstruction for totally laparoscopic colectomies.

Characterization of FsXEG12A from the cellulose-degrading ectosymbiotic fungus Fusarium spp. strain EI cultured by the ambrosia beetle.

Despite the threat of Fusarium dieback posed due to ambrosia fungi cultured by ambrosia beetles such as Euwallacea spp., the wood-degradation mechanisms utilized by ambrosia fungi are not fully understood. In this study, we analyzed the 16S rRNA and 18S rRNA genes of the microbial community from the Ficus tree tunnel excavated by Euwallacea interjectus and isolated the cellulose-degrading fungus, Fusarium spp. strain EI, by enrichment culture with carboxymethyl cellulose as the sole carbon source. The cellulolytic enzyme secreted by the fungus was identified and expressed in Pichia pastoris, and its enzymatic properties were characterized. The cellulolytic enzyme, termed FsXEG12A, could hydrolyze carboxymethyl cellulose, microcrystalline cellulose, xyloglucan, lichenan, and glucomannan, indicating that the broad substrate specificity of FsXEG12A could be beneficial for degrading complex wood components such as cellulose, xyloglucan, and galactoglucomannan in angiosperms. Inhibition of FsXEG12A function is, thus, an effective target for Fusarium dieback caused by Euwallacea spp.

Laparoscopic inguinal hernia repair with a joystick-guided robotic scope holder (Soloassist II®): retrospective comparative study with human assistant.

PURPOSE: The purpose of this study was to evaluate the clinical usefulness of a joystick-guided robotic scope holder (Soloassist II®) in laparoscopic inguinal hernia repair. METHODS: Among 182 inguinal hernia patients treated by laparoscopic transabdominal preperitoneal repair, 82 cases were completed with a human scope assistant, while Soloassist was used in 100 cases. We retrospectively compared perioperative results of Soloassist group and human scope assistant group. In 139 unilateral cases, we also used logistic regression of perioperative factors for the propensity score calculation to balance the bias. RESULTS: All operations with Soloassist were carried out laparoscopically as solo-surgery without any system-specific complications. A statistically significant decrease in operation time was observed in Soloassist group compared with human assistant group (93.6 vs 85.9 min, p = 0.05). There was no prolongation of preoperative time or difference in the amount of intraoperative blood loss. Operation time was also significantly shorter in Soloassist group, when analyzing unilateral cases (85.5 vs 76.3 min, p = 0.02) and bilateral cases (126.9 vs 111.8 min, p = 0.01), independently. However, after propensity score matching in unilateral cases, there was no statistically significant difference between the two groups (83.8 vs 77.2 min, p = 0.23). CONCLUSIONS: The feasibility of Soloassist in laparoscopic inguinal hernia repair was demonstrated with no adverse device-related events. All surgeries could be completed as solo-surgery, while no additional time for preoperative setting was required. The mean operation time tends to be shorter in Soloassist group compared with human assistant group. Soloassist could be an effective device in laparoscopic inguinal hernia repair.

Comparative effectiveness of human scope assistant versus robotic scope holder in laparoscopic resection for colorectal cancer.

BACKGROUND: Several types of robotic scope holders have been developed to date, but there are only some experimental reports or the results of small clinical cases. The Soloassist® system is a unique robotic scope holder with which the surgeon can control the field of view by a joystick. We evaluated the efficacy of Soloassist in laparoscopic resection for colorectal cancer. MATERIALS AND METHODS: We investigated operative time, blood loss, setup time, length of hospital stay, and the number of participating surgeons in 273 laparoscopic colorectal resections, including 130 cases with human assistant (HA group) and 143 cases with Soloassist (SA group). Additionally, we also used logistic regression of the perioperative factors for the propensity score calculation to balance the bias. RESULTS: The number of participating surgeons was apparently less in the SA group (HA group: 3.3 vs. SA group: 2.5, p < 0.01). The average operative time was shorter in the SA group, but there was no statistical difference (HA group: 287.0 min vs. SA group: 268.5 min, p = 0.07). No significant difference was found in setup time, conversion rate, perioperative complications, and length of hospital stay. There was no conversion case to human scope assistant and no system-specific adverse event. Similar results were observed between two groups after propensity score matching. CONCLUSION: Laparoscopic colorectal resection with Soloassist is safe and feasible. The present study demonstrated that Soloassist system provided the possibilities of saving human resources in laparoscopic colorectal resection without prolonged operative time or system-specific morbidity. Soloassist is an effective robot-assisted surgical instrument for colorectal surgery.

Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi.

Pectinolytic enzymes are used in diverse industrial applications. We sought to isolate a pectate lyase from Aspergillus luchuensis var. saitoi, a filamentous fungus used in traditional food and beverage preparation in Japan. The identified enzyme, named AsPelA, is orthologous to PelA from A. luchuensis mut. kawachii (AkPelA); the enzymes exhibit 99% amino acid sequence identity, with Ile140 and Val197 of AsPelA being replaced by Val and Asp in AkPelA, respectively. AsPelA activity decreased to 71%, 61%, and 46% of maximal activity after 60-min incubation at 60 °C, 70 °C, and 80 °C, whereas AkPelA activity dropped to 16%, 10%, and 8.5%, respectively, indicating that AsPelA is more thermostable than AkPelA. Furthermore, AsPelA was stable within a neutral-to-alkaline pH range, as well as in the presence of organic solvents, detergents, and metal ions. Our findings suggest that AsPelA represents a candidate pectate lyase for applications in food, paper, and textile industries.

Feasibility and Usefulness of a Joystick-Guided Robotic Scope Holder (Soloassist) in Laparoscopic Surgery.

INTRODUCTION: The Soloassist® system is a joystick-guided robotic scope holder. We evaluated the efficacy of Soloassist in laparoscopic surgery. METHODS: We investigated operative time, blood loss, set-up time, length of hospital stay, and the number of participating surgeons in laparoscopic cholecystectomy cases before and after the introduction of Soloassist. Furthermore, we evaluated these factors in each group of 20 elective and emergency cholecystectomy cases by single surgeon after matching their background. To evaluate the performance level of operating Soloassist, we divided the operative field into three areas. Then we counted the frequency of energy device activation in initially 10 cases by a single surgical resident and observed its change. RESULTS: The number of participating surgeons was significantly less and postoperative hospital days were fewer in the Soloassist group. There was no significant difference between set-up time and blood loss both in elective and emergency cases. The total number of energy device activations and that in the dangerous area decreased in accordance with the experience. CONCLUSION: Considering our results and previous reports, the combination use of an ideal active scope holder and a commercially available 3D scope is currently considered the best approach in laparoscopic surgery. In the near future, development of active scope holders might play an important role in laparoscopic surgery.

Ileal schwannoma developing into ileocolic intussusception.

Intussusception is rare in adults. We describe a 47-year-old man with ileal schwannoma that led to ileocolic intussusception. Abdominal ultrasonography, abdominal CT scan and barium enema confirmed an ileal tumor. Colonoscopy revealed a peduncular submucosal tumor (SMT) 75 mm long with an ulcerated apex at the ascending colon. The provisional diagnosis was a gastrointestinal stromal tumor of the terminal ileum. Ileocecal resection was carried out and the tumor was histologically diagnosed as schwannoma. Abdominal pain resolved postoperatively. This case reminds us that ileal schwannoma should be included in the differential diagnosis of intussusception caused by an SMT in the intestine.

The association between RAD18 Arg302Gln polymorphism and the risk of human non-small-cell lung cancer.

PURPOSE: The repair enzyme RAD18 plays a key role in the post-replication repair process in various organisms from yeast to human, and the molecular function of the RAD18 protein has been elucidated. Single nucleotide polymorphism (SNP) of arginine (Arg, CGA) or glutamine (Gln, CAA) at codon 302 is known on RAD18; however, the association between the SNP and the risk of any human cancers including non-small-cell lung cancer (NSCLC) has not been reported. We therefore investigated the relationship between the polymorphism and the development and progression of human NSCLC. METHODS: The study population included 159 patients with NSCLC and 200 healthy controls. The SNP was genotyped by polymerase chain reaction with the confronting two-pair primer (PCR-CTPP) assay. Genotype frequencies were compared between patients and controls, and the association of genotypes with clinicopathological parameters was also studied. RESULTS: The Gln/Gln genotype was significantly more frequent in NSCLC patients (20.7%) than in healthy controls (11.5%)(P = 0.003). The increased risk was detected in NSCLC patients with the Gln/Gln genotype [Odds ratio (OR) = 2.63, 95% confidence interval (CI)=1.38-4.98]. As to the relationship of the SNP with clinicopathological parameters of NSCLC, significantly higher risks were detected in lung squamous cell carcinoma (LSC) (OR = 4.40, 95% CI = 1.60-12.1). CONCLUSIONS: Our results suggested that Gln/Gln genotype of the RAD18 SNP has the increased risk of NSCLC, especially of LSC. This is the first report to provide evidence for an association between the RAD18 Arg302Gln polymorphism and human NSCLC risk.

Decreased expression of the SIN3A gene, a candidate tumor suppressor located at the prevalent allelic loss region 15q23 in non-small cell lung cancer.

To identify the tumor suppressor genes (TSG) associated with non-small cell lung cancers (NSCLC), we performed the loss of heterozygosity (LOH) analysis in NSCLC samples from 66 patients. We focused on the novel hot spot region on 15q14-24 with eight polymorphic microsatellite markers. Frequent allelic loss was detected in 33 of 48 informative cases (69%) at D15S984 on 15q23. We defined the fine map on the region and identified the SIN3A gene as a candidate TSG. The SIN3A gene product is a component of the histone deacetylase (HDAC) complex and plays essential roles in early embryonic development and the proliferation and survival of a variety of cells through the repression of diverse signaling pathways. Our expression analysis revealed more frequent down-regulation of the SIN3A mRNA in 19 of 31 cases (61%) of NSCLCs in comparison to those of other flanking genes (16-42%), albeit the correlation of the decreased expression with the LOH did not attain statistic significance. These results suggest that the attenuated function of SIN3A due to a decreased level of expression may result in epigenetic de-regulation of growth-related genes through histone acetylation, which leads to the tumorigenesis of lung cancer cells. To our knowledge, this is the first evidence of the down-regulation of the SIN3A gene in human cancer.

Single nucleotide polymorphism in the RAD18 gene and risk of colorectal cancer in the Japanese population.

The RAD18 gene, located on the human chromosome 3p24-p25, plays a crucial role in post-replication repair (PRR) in various organisms from yeast to humans. In the human RAD18 gene, one coding single nucleotide polymorphism (SNP) at codon 302, encoding either arginine (Arg, CGA) or glutamine (Gln, CAA), was reported. Although the molecular function of the RAD18 protein came to be elucidated, the association between the RAD18 Arg302Gln polymorphism and the risk of human cancer development was not examined. Therefore, we investigated the relationship between the polymorphism and the development of human primary colorectal cancer (CRC). The Arg302Gln polymorphism in 100 patients with CRC and 200 healthy controls were genotyped by the polymerase chain reaction with confronting two-pair primer (PCR-CTPP) assay. The Gln/Gln genotype was significantly more frequent in CRC (18.0%) than in the healthy controls (11.5%) (p=0.046). The increased risk was detected in CRC patients with the Gln/Gln genotype (Odds ratio [OR], 2.10; 95% confidence interval [CI], 1.00 to 4.40). When the relationship of the SNP with clinicopathological parameters of CRC was investigated, particularly in the well-differentiated grade and in the lymph node metastasis (N1) CRC patients, significantly higher risks were detected (OR, 7.00; 95% CI, 1.19-41.1 and OR, 3.71; 95% CI, 1.30-10.6, respectively). These results suggested that the RAD18 Arg302Gln polymorphism is associated with the risk of CRC. This report provides evidence for an association between the RAD18 Arg302Gln polymorphism and human CRC risk.

Single nucleotide polymorphism of the AXIN2 gene is preferentially associated with human lung cancer risk in a Japanese population.

The AXIN2 gene, a negative regulator gene of Wnt/beta-catenin signaling, is a putative tumor suppressor gene on human chromosome 17q24. In the genomic locus on which the AXIN2 gene is located, allelic loss and rearrangement were frequently detected in many cancers. An association between human cancer risk and a single nucleotide polymorphism (SNP) at codon 50 of the AXIN2 gene, encoding either proline (CCT) or serine (TCT), remains undefined. We, therefore, investigated the distribution of the SNP at codon 50 in 110 healthy controls and 160 patients with non-small-cell lung cancer, 113 patients with colorectal cancer, and 63 patients with head and neck cancer. We found that the frequency of the homozygous T/T (Ser/Ser) genotype was significantly less in lung cancer patients (5.0%) than in healthy controls (13.6%) (p=0.005). As compared with the C/C (Pro/Pro) genotype of the controls, lung cancer patients with the T/T genotype showed reduced risk of cancer; the adjusted odds ratio (OR) for patients with the homozygous T/T (Ser/Ser) genotype was 0.31 (95% confidence interval (CI), 0.12-0.79). The association was particularly strong in lung cancer patients with lung adenocarcinoma (LAD) (adjusted OR, 0.24; 95% CI, 0.07-0.81), with well-differentiated grade cancer (adjusted OR, 0.12; 95% CI, 0.01-0.99) and with moderately-differentiated grade cancer (adjusted OR, 0.18; 95% CI, 0.04-0.85). These results suggest that the AXIN2 Pro50Ser SNP is associated with development of lung cancer as a protective SNP, while an association between the AXIN2 SNP and risk of colorectal cancer and of head and neck cancer was not observed. This is the first report to show an association between the AXIN2 SNP and lung cancer risk.

Single nucleotide polymorphisms in the EXO1 gene and risk of colorectal cancer in a Japanese population.

EXO1 is a member of the RAD2 nuclease family and functions in DNA replication, repair and recombination. We investigated the relationship of single nucleotide polymorphisms (SNPs) at exon 10 (T439M) and exon 13 (P757L) of the EXO1 gene with development, progression and metastasis of colorectal cancer. For T439M, the Thr/Met genotype [odds ratio (OR) = 2.03, 95% confidence interval (CI) 1.04-3.98] and Thr/Met and Met/Met genotypes combined (OR = 2.37, 95% CI 1.23-4.56) demonstrated significant association with the development of colorectal cancer after adjusting for age, gender and smoking status. For P757L, patients with the Leu/Leu genotype showed a reduced risk of colorectal cancer (adjusted OR = 0.398, 95% CI 0.183-0.866) when the Pro/Leu and Pro/Pro genotypes were combined and used as the reference. The Leu/Leu genotype also had a reduced risk (adjusted OR = 0.373, 95% CI 0.164-0.850) when the Pro/Leu genotype was used as the reference. Individuals who carried both putative risk genotypes (Thr/Met and Met/Met for T439M and Pro/Leu for P757L) showed an adjusted OR of 4.95 (95% CI 1.56-15.7) compared with those who carried both low risk genotypes. Analysis of microsatellite instability (MSI) revealed that tumors from individuals who carried both putative risk genotypes tended to have a higher frequency of MSI positives than those from patients who carried both low risk genotypes, although a significant correlation was not found between EXO1 genotype and MSI status. This is the first report to provide evidence for an association of EXO1 gene polymorphisms with colorectal cancer risk. The EXO1 genotypes were not associated with any clinicopathological characteristics in colorectal cancer patients.

Modulation by hyperpolarization-activated cationic currents of voltage responses in human rods.

We used the whole-cell patch-clamp recording technique on surgically excised human retina to examine whether human rod photoreceptors express hyperpolarization-activated cationic currents (I(h)) and to analyze the effects of I(h) on rod's voltage responses. Hyperpolarizing voltage steps from a holding potential of -60 mV evoked a slow inward-rectifying current in both rods in retinal slices and isolated rods. The slow inward-rectifying currents induced by hyperpolarization were markedly reduced by 3 mM Cs(+) (a blocker of I(h)) in the bath, but not by 3 mM Ba(2+) (an anomalous rectifier K(+) current blocker) or 1 mM SITS (a Cl(-) current blocker). A concentration-response curve for block by Cs(+) of the inward currents could be fitted by the Hill equation with a half-blocking concentration (IC(50)) of 41 microM and a Hill coefficient of 0.91. The time course of the inward current activation was well described at all recorded voltages by the sum of two exponentials. Under current-clamp conditions, injection of steps of current, either hyperpolarizing or depolarizing, elicited an initial rapid voltage change that was followed by a gradual decay in the voltage response. The decay in the voltage responses was eliminated by bath application of 3 mM Cs(+). The voltage dependence, pharmacology, and kinetics of the slow inward-rectifying currents described above suggest that human rods express I(h). We suggest that I(h) becomes activated in the course of large hyperpolarizations generated by bright-light illumination and may modify the waveform of the photovoltage in human rods.

Transparent irrigation cannula for bimanual lens cortex removal.

A transparent irrigation cannula was developed to facilitate bimanual removal of subincisional lens cortex after phacoemulsification. The cannula allows visualization of the proximal cortex and provides ample inflow to maintain an inflated anterior chamber even at a high aspiration pressure.