A novel nonsecosteroidal VDR agonist (CH5036249) exhibits efficacy in a spontaneous benign prostatic hyperplasia beagle model.

To date, there have been no reports showing the efficacy of nonsecosteroidal vitamin D receptor (VDR) agonists in a benign prostatic hyperplasia (BPH) animal model. To examine the efficacy of CH5036249, a novel nonsecosteroidal VDR agonist, we orally administered the compound at 0.03 microg/kg to a beagle model with spontaneous BPH. Prostate volume was checked by rectal ultrasonic probe periodically during 11 months of administration and the prostate tissues histologically examined. CH5036249 inhibited prostate growth in two out of three dogs compared with vehicle-treated dogs. In the prostate specimens, substantial atrophy of the epithelium was observed in all dogs administered CH5036249. At the dose given, serum calcium levels slightly increased in the CH5036249-treated dogs but stayed within a normal range. We next examined the cell growth inhibition of CH5036249 using human prostate stromal cells and found the cell growth inhibitory activity of CH5036249 to be comparable to that of 1alpha,25(OH)2D3. The bioavailability from oral administration in rats was 95.1% with a t1/2 of 17.6 h. Both micro-AMES and micronucleus tests were negative. Although the results are still preliminary, we consider the novel nonsecosteroidal VDR agonist CH5036249 to be a possible new drug candidate for the treatment of BPH in humans.

Irreversible morphological changes in leg bone following chronic gravitational unloading of growing rats.

Effects of gravitational unloading or loading on the growth and development of hindlimb bones were studied in rats. Male Wistar rats were hindlimb-unloaded or loaded at 2-G from the postnatal day 4 to month 3. The morphology and mineral content of tibia and fibula, as well as the mobility of ankle joints, were measured at the end of 3-month suspension or loading, and 1, 2, and 3 months after ambulation recovery. Growth-related increases of bone weight and mineral density were inhibited by unloading. But they were gradually recovered toward the control levels, even though they were still less than those in the age-matched controls after 3 months. None of the parameters were influenced by 2-G loading. However, here we report that chronic unloading causes abnormal morphological development in hindlimb bone of growing rats. Irreversible external bend of the shaft and rotation of the distal end of tibia, which limit the dorsiflexion of ankle joints, were induced following chronic gravitational unloading during developing period. It is also suggested that such phenomena are caused by the abnormal mechanical forces imposed by muscle utilization with altered patterns. The activity of ankle dorsiflexor was increased and that of plantarflexor was inhibited during unloading.

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Mechanical load-dependent regulation of satellite cell and fiber size in rat soleus muscle.

The effects of mechanical unloading and reloading on the properties of rat soleus muscle fibers were investigated in male Wistar Hannover rats. Satellite cells in the fibers of control rats were distributed evenly throughout the fiber length. After 16 days of hindlimb unloading, the number of satellite cells in the central, but not the proximal or distal, region of the fiber was decreased. The number of satellite cells in the central region gradually increased during the 16-day period of reloading. The mean sarcomere length in the central region of the fibers was passively shortened during unloading due to the plantarflexed position at the ankle joint: sarcomere length was maintained at <2.1 microm, which is a critical length for tension development. Myonuclear number and domain size, fiber cross-sectional area, and the total number of mitotically active and quiescent satellite cells of whole muscle fibers were lower than control fibers after 16 days of unloading. These values then returned to control values after 16 days of reloading. These results suggest that satellite cells play an important role in the regulation of muscle fiber properties. The data also indicate that the satellite cell-related regulation of muscle fiber properties is dependent on the level of mechanical loading, which, in turn, is influenced by the mean sarcomere length. However, it is still unclear why the region-specific responses, which were obvious in satellite cells, were not induced in myonuclear number and fiber cross-sectional area.

[Intrathoracic bleeding during video-assisted thoracoscopic lobectomy and segmentectomy].

We have reviewed our experience from January 2001 through January 2003 in 33 video-assisted thoracoscopic lobectomy and segmentectomy (VATS) in patients with cT1N0M0 lung cancer to look at intraoperative bleeding from pulmonary vessels. Intraoperative bleeding occurred in 15 cases, 45.5% of 33 VATS procedures, and 2 cases, 6.1% of VATS procedures converted to an open procedure. Intraoperative bleeding occurred more frequently in VATS segmentectomy than VATS lobectomy. Most of bleeding from pulmonary arteries and veins can be controlled by compression, and they can be controlled thoracoscopically by tie or suture through the utility thoracotomy. But, significant bleeding from pulmonary arteries, which can not be controlled with a mounted swab, it should be converted to an open procedure.

Transport characteristics of peptides and peptidomimetics: I. N-methylated peptides as substrates for the oligopeptide transporter and P-glycoprotein in the intestinal mucosa.

Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N-Methylation has been used successfully in peptide-based drug design in an attempt to improve the metabolic stability of a peptide-based lead compound. However, the effect of N-methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P-glycoprotein (P-gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N-acetylated and C-amidated) analogs of H-Ala-Phe-Ala-OH with N-methyl groups on either the Ala-Phe or Phe-Ala peptide bond were synthesized. Transport studies using Caco-2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N-methylation of the Ala-Phe peptide bond of H-Ala-Phe-Ala-OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N-methylation of the Phe-Ala peptide bond of H-Ala-Phe-Ala-OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N-methylation of the Phe-Ala peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 decreased the substrate activity of the molecule for the efflux transporter P-gp. In contrast, N-methylation of the Ala-Phe peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 increased the substrate activity of the molecule for P-gp.

Isolation and expression of a gene (CGR1) regulated during the yeast-hyphal transition in Candida albicans.

We used RNA fingerprinting of arbitrarily primed PCR to isolate genes upregulated during the yeast-hyphal transition in Candida albicans. The sequence and expression of one of these genes (CGR1, Candida growth regulation) are presented. Our results suggest that CGR1 expression is associated with a growth cessation of yeast cells, a prerequisite for germination in this organism.

Stabilizing and solubilizing effects of sulfobutyl ether beta-cyclodextrin on prostaglandin E1 analogue.

PURPOSE: Parent cyclodextrins are known to accelerate the degradations such as dehydration and isomerization of E-type prostaglandins in neutral and alkaline solutions. The objective of this study was to attempt the stabilization and solubilization of E1-type prostaglandin analogue in aqueous solution by biocompatible cyclodextrin derivatives. METHODS: The interaction of an E1-type prostaglandin, methyl 7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxy-4-(m-methoxymethylphenyl)1-butenyl]-5-oxocyclopentyl]-5-thiaheptanoate (MEester) with cyclodextrins (CyDs) was studied by spectroscopies and the solubility method. The degradation of MEester was monitored by high-performance liquid chromatography. RESULTS: 1H-nuclear magnetic resonance spectroscopic studies indicated that MEester forms 1:1 inclusion complexes with alpha-, beta-, and gamma-CyDs in solutions, where alpha-CyD interacts with the a-side chain containing methyl ester moiety of the drug, whereas beta- and gamma-CyDs preferentially include around the five-membered ring and both side chains of the drug. Parent alpha-CyD and hydrophilic derivatives, such as 2-hydoxypropyl-alpha- and -beta-CyDs, sulfobutyl ether beta-CyD (SBE-beta-CyD) and maltosyl beta-CyD showed higher solubilizing abilities against MEester over parent beta- and gamma-CyDs. SBE-beta-CyD and 2,6-dimethyl-beta-CyD (DM-beta-CyD) significantly decelerated the degradation of MEester, particularly the base-catalyzed dehydration, in neutral and alkaline solutions, whereas other CyDs accelerated the degradation. The acid-catalyzed degradation of MEester (pH < 3) was decelerated by the addition of CyDs, especially alpha-CyD. CONCLUSIONS: SBE-beta-CyD with low hemolytic activity and low toxicity is useful as a pharmaceutical carrier for the preparation of injectable MEester, because of its higher stabilizing and solubilizing effects on MEester. Furthermore, SBE-beta-CyD can be useful as a stabilizing agent for drugs, that are subject to base-catalyzed degradations, probably because of the electric repulsion between anionic charges of the sulfobutyl moiety and catalytic anionic species such as hydroxide ion.

[Ocular counter-rolling after prolonged alteration in the direction of gravity].

To investigate the dynamics of otolith and oculomotor function, we subjected volunteers to a lateral body tilt for a period of two hours and analyzed ocular counter-rolling. Six healthy adult volunteers were roll-tilted along the naso-occipital axis at a speed of 0 degree/sec. from the 0 degree earth vertical position to the 90 degrees lateral tilt position. After two hours, the volunteers were returned to the 0 degree earth vertical position. Ocular counter-rolling was recorded using an infrared CCD camera. The video recordings were made in the dark while the volunteers had their eyes open. Recordings were obtained (1) as the volunteers was moved from the 0 degree earth vertical position to the 90 degrees lateral position, (2) 5 minutes after reaching the lateral position, (3) during the roll-back from the 90 degrees lateral position to the 0 degree upright position, two hours after the lateral tilt loading, and (4) 5 minutes after reaching the final upright position. The occurrence of ocular counter-rolling between the 0 degree upright position and the 30 degrees tilt position was confirmed during both roll movements from 0 degree to 90 degrees and from 90 degrees to 0 degree. The counter-rolling was most noticeable between the 0 degree and the 30 degrees positions. No differences in ocular counter-rolling during the roll-tilt and the roll-back situations were observed. These results indicate that the two-hour lateral tilt position did not produce any functional changes in the hair cells and/or the otolith-oculomotor system. All of the subjects exhibited ocular counter-counter-rolling during the initial stage of their roll-back to a normal upright position. This phenomenon might result from the additional bending of the hairs by inertia during the initiation of the backward roll motion.

The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase.

We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation. The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae. The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S. cerevisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system. We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening.

Identification of a novel inhibitor specific to the fungal chitin synthase. Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans.

As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K(i) value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC(50) value of 0.27 microm. In the presence of RO-09-3143, the C. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2Delta null mutants but not in the cachs3Delta null mutants. Thus, it appears that in C. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.

Synthesis and structure-activity relationships of novel fungal chitin synthase inhibitors.

A novel Candida albicans chitin synthase 1 (CaChs1) inhibitor, RO-41-0986 (1) was discovered by random screening. Systematic modification led to the identification of a highly potent CaChs1 inhibitor, RO-09-3024 (2), having strong antifungal activity against Candida spp. in vitro.

Vasomotor sympathetic nerve activity in men during bed rest and on orthostasis after bed rest.

BACKGROUND: Alterations in autonomic function are commonly seen during and after spaceflight, and its ground-based analog, 6 degrees head-down bed rest (HDBR). They may include peripheral vascular regulation, but vasomotor sympathetic efferent nerve discharges to peripheral vasculatures have not been examined. The aim of our study was to examine changes in vasomotor sympathetic nerve activity during HDBR and under orthostasis after HDBR. METHODS: We performed 6 d of HDBR on six male subjects, and measured muscle sympathetic nerve activity (MSNA) together with plasma norepinephrine concentrations in the supine position before HDBR and in 6 degrees head-down position on the sixth day (HDBR6) of HDBR. We also measured MSNA in head-up tilt (HUT) test before and after HDBR. RESULTS: On HDBR6, MSNA burst rate was the same (17+/-4 bursts x min(-1)) as that in supine position before HDBR (15+/-2 bursts min(-1)), but plasma norepinephrine concentrations were decreased to 1.14+/-0.10 pmol x ml(-1) compared with the supine value before HDBR (1.56+/-0.20 pmol x ml(-1), p<0.05). After HDBR, supine MSNA burst rate significantly increased by 58% to 24+/-4 bursts x min(-1). MSNA increment in response to HUT was similar between before (34+/-3 bursts min(-1) x sin HUT(-1)) and after (40+/-6 bursts x min(-1) x sin HUT(-1)) HDBR. CONCLUSIONS: Our findings suggest that: a) the relationship between MSNA and plasma norepinephrine concentrations was altered on the sixth day during HDBR; b) the vasomotor sympathetic nerve activity was enhanced after HDBR; and c) the augmentation of vasomotor sympathetic outflow to muscles under orthostasis was preserved after HDBR.

Candida albicans: adherence, signaling and virulence.

The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to filamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cerevisiae pseudohyphal/mating pathway, which utilizes the regulatory protein, Cphlp. The other pathway is not totally defined but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p/Nik1p. Mutations in strains, which specifically target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently defined whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a filamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been defined. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron).

[Coronary artery bypass grafting for a patient with angina pectoris and ulcerative colitis].

The implementation of coronary artery bypass grafting for angina pectoris with ulcerative colitis has been rarely reported. A 63-year-old man has a past history of acute myocardial infarction in 1984 and ulcerative colitis since 1988. Coronary angiography and cardiac catheterization showed total obstruction of segment 2, 95% stenosis of segment 6, 75% stenosis of segment 7 and total obstruction of segment 12 with LVEF 23%. Coronary artery bypass grafting was performed under IABP support and cardiopulmonary bypass with aprotinin infusion after an inflammatory reaction of ulcerative colitis was adequately suppressed. Ulcerative colitis was controlled by administering 40 mg of predonisolone during perioperative period.

Changes in body fluid distribution during 7 days 6 degrees head-down bed rest.

It is well known that, in a state of weightlessness, body fluids shift toward the head site, but the actual rates of the migration of fluids has never been measured. In order to identify the causes of reduced orthostatic tolerance and the reduction in circulating blood volume after returning from a space mission, it is necessary to measure the changes in body fluid distribution. Although the methods for measuring changes in body fluids include the use of isotopes or pigments, such methods cannot be employed in space due to their invasive properties. Therefore, we directed our attention toward the impedance method, for which we developed a non-invasive apparatus for measuring the distribution of body fluids. We therefore measured the actual distribution of body fluids using the bed-rest method, a model of the zero gravity state.

Neuroprotective effect of YM-39558, orotic acid ethylester, in gerbil forebrain ischemia.

We studied the effects of orotic acid and YM-39558 (2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid ethyl ester), orotic acid ethylester, on delayed neuronal death of hippocampal CA1 neurons induced by transient forebrain ischemia. Our data indicated that YM-39558 had high permeability across the blood brain barrier and was hydrolyzed to orotic acid, the active substance, in the brain. The neuronal damage was reduced significantly in animals intraperitoneally treated with YM-39558 (100 mg/kg x 3) after ischemia, but not with orotic acid in the same way. The results also suggested that the maintenance of a few ten micromolar orotic acid in cerebrospinal fluid were needed for its neuroprotective effects.

Transport characteristics of peptidomimetics. Effect of the pyrrolinone bioisostere on transport across Caco-2 cell monolayers.

PURPOSE: To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. METHODS: Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. RESULTS: In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (Papp) calculated for the BL-to-AP and AP-to-BL transport (P(BL-->AP)/P(AP-->BL)) varied between 1.7 and 36.2. When individual pairs were ompared, P(BL-->AP)/P(AP-BL) ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 microM cyclosporin A to the transport buffer reduced the P(BL-->AP)/P(AP-->BL) ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 microM cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. CONCLUSIONS: The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.

Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae. Identification of additional amino acid residues involved in its catalytic activity.

Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p. Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme. Here we report that another region, con2, in the C-terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S. cerevisiae Chs1p and Chs2p. Alanine substitutions for the conserved amino acids in con2 identified five amino acids, Asn797, His799, Asp800, Trp803, and Thr805, the mutation of which severely diminished enzymatic activity and the enzyme's ability to rescue the yeast chs2 delta chs3 delta null mutant strain. Although the activities of some of the mutant enzymes were too low to measure enzyme kinetics, most of the alanine mutations in con2 affected the kcat values rather than the K(m) values. Whereas a conservative mutation of Asn797 restored the activity, those of His799, Asp800, Trp803, and Thr805 did not. A fine alignment of the amino acid sequences of con2 and Chs3p revealed that Asp800, Trp803 and Thr805 are completely conserved near the C-terminal ends of Chs3p and its homologs in other fungi. On the basis of these findings, we propose that Asp800, Trp803, and Thr805 in con2 are additional residues involved in catalysis, and hypothesise that Asp800 together with the previously identified Asp441 and Asp562 serve as polar residues necessary for the acid-based catalytic reaction of chitin synthase.

Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth.

A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid. The biochemical characteristics of chitin synthase B (ChsB) expressed in S. cerevisiae were examined. The chitin synthase B produced in galactose medium showed zymogenicity due to activation by trypsin treatment and required Mg2+ ion to exert maximal activity. It was competitively inhibited by polyoxin D. The Ki value of the inhibitor was 10 microM, and the K(m) for the substrate was 1.6 mM. The activity was enhanced by the addition of N-acetylglucosamine. The optimal pH is 7.5 when Mg2+ is used. These characteristics are the same as those of other chitin synthases.