The Effect of Japanese Herbal Medicines (Kampo) Goreisan and Saireito on the Prevention of Recurrent Chronic Subdural Hematoma: A Prospective Randomized Study.

BACKGROUND AND OBJECTIVES: Chronic subdural hematoma (CSDH) sometimes recurs after surgical treatment and requires reoperation. In Japan, Japanese herbal medicines (Kampo), such as Goreisan and Saireito, have been used as adjunctive therapies to prevent the recurrence of CSDH. However, no prospective randomized study has proven the efficacy of Kampo medicine in all patients. To investigate whether Goreisan and Saireito reduce the postoperative recurrence of CSDH in a prospective randomized study. METHODS: Between April 2017 and July 2019, a total of 118 patients who underwent initial burr hole surgery for CSDH were randomly assigned to the following 3 groups: (1) Goreisan for 3 months (Group G), (2) Saireito for 3 months (Group S), and (3) no medication (Group N). The primary end point was symptomatic recurrence within 3 months postoperatively, and the secondary end point was complications associated with the administration of Kampo medicine. RESULTS: Among 118 patients, 114 (Group N, n = 39; Group G, n = 37; and Group S, n = 38) were included in our analysis. In this study, byakujutsu (containing Atractylodes rhizome ) Goreisan and Saireito were used, unlike other prospective randomized studies in which sojutsu (containing Atractylodes lancea rhizome) Goreisan was used. The overall recurrence rate was 11.4% (13/114: 10 for Group N, 2 for Group G, and 1 for Group S). The recurrence rate of Group G was significantly lower than that of Group N (5.4% vs 25.6%; P = .043). The recurrence rate of Group S was also significantly lower than that of Group N (2.6% vs 25.6%; P = .02). No patients developed complications associated with the administration of Kampo medicine. CONCLUSION: This is the first study to show that Kampo medicine reduced the recurrence rate of CSDH in an overall population. This study demonstrated that byakujutsu Goreisan and Saireito may have favorable effects, unlike other studies, because byakujutsu has stronger anti-inflammatory activity than sojutsu.

Cervical radiculopathy due to extracranial vertebral artery dissection treated by stent placement with a flow diversion effect: illustrative case.

BACKGROUND: Cervical radiculopathy due to extracranial vertebral artery dissection (VAD) is extremely rare. The disease is usually treated with conservative treatment because of its favorable prognosis. However, there is a possibility that conservative treatment may bring about no improvement in radiculopathy. Although stent placement with a flow diversion effect may be effective in such cases, there are no reported cases that were treated with stent placement. OBSERVATIONS: A 40-year-old healthy man presented with severe right neck pain, right arm pain, and right arm weakness after cracking his neck. A neurological examination revealed right C5 radiculopathy. Neuroimaging studies revealed right extracranial VAD. The VAD compressed the right C5 nerve root. Although medications were administered, there was no improvement in the symptoms. He experienced severe radicular pain. The authors performed stent placement with a flow diversion effect 10 days after the onset of VAD. His radicular pain improved immediately after the procedure, and the remaining radiculopathy completely improved within 1 month. Follow-up angiography showed complete improvement of the VAD. LESSONS: Stent placement with a flow diversion effect may be considered when radiculopathy that hinders a patient's daily life exists. Stent placement may bring about rapid improvement in radiculopathy, especially radicular pain.

Postoperative D-dimer levels predict venous thromboembolisms detected with contrast-enhanced computerized tomography in patients undergoing anterior cruciate ligament reconstruction.

BACKGROUND: In the literature, factors associated with postoperative venous thromboembolisms (VTEs) after anterior cruciate ligament reconstruction (ACLR) are limited. This study aimed to investigate the incidence of venous thromboembolisms (VTEs) after anterior cruciate ligament reconstruction (ACLR) and to identify risk and predictive factors for VTEs. METHODS: This retrospective study included 136 patients who underwent arthroscopic ACLR with mechanical prophylaxis between April 2012 and July 2022. Contrast-enhanced computed tomography (CT) was applied to detect VTEs comprising deep venous thromboses and pulmonary embolisms 7 days after surgery. Data including age, sex, body mass index, concomitant treatments, graft types, smoking status, operative and tourniquet times, postoperative D-dimer levels, and other laboratory test results, were collected for analyses. The incidence of radiographically confirmed VTEs and the associated risk factors, such as age, sex, body mass index, concomitant treatments, graft types, smoking status, operative and tourniquet times, postoperative D-dimer levels, and other laboratory test results, were analyzed. RESULTS: The overall incidence of radiographic VTEs was 11.0% (15 cases) in 136 patients. There was one symptomatic patient who had Homan's sign. Multivariable analysis indicated that postoperative D-dimer level was an independent factor related to a radiographic VTE after ACLR, although there was no association between radiographic VTEs and preoperative status or operation status. The optimal cutoff value for postoperative D-dimer level was 2.8 µg/ml according to the receiver operating characteristic curve analysis, with a sensitivity of 80.0% and specificity of 83.5%. CONCLUSION: The incidence of ACLR-associated radiographical VTEs (deep venous thrombosis and pulmonary embolism) under mechanical prophylaxis was 11.0% in this study. An elevated D-dimer level at 7 days after surgery is an independent predictor of VTE in patients undergoing ACLR. The postoperative D-dimer level is a more reliable marker for identifying VTE in patients who underwent ACLR.

High yield production of norovirus GII.4 virus-like particles using silkworm pupae and evaluation of their protective immunogenicity.

Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9 mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40 nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS.

Polarization-Type Potential-Induced Degradation in Front-Emitter p-Type and n-Type Crystalline Silicon Solar Cells.

For SiO2 layers underneath the SiN x antireflection/passivation layers of front-emitter p-type c-Si solar cells, this paper presents an investigation into their effects on polarization-type potential-induced degradation (PID), in addition to a comparison of polarization-type PID behavior in front-emitter p-type c-Si cells and front-emitter n-type c-Si cells. After PID tests with a bias of +1000 V, p-type c-Si cells without SiO2 layers underneath the SiN x layers showed no degradation, although p-type c-Si cells with approx. 10 nm thick SiO2 layers showed polarization-type PID, which is characterized by a reduction of the short-circuit current density and the open-circuit voltage. This result implies that highly insulating layers such as SiO2 layers play an important role in the occurrence of polarization-type PID. Comparison of polarization-type PID in p-type and n-type c-Si cells with SiO2 layers indicated that degradation in the n-type cells is greater and saturates in a shorter time than in the p-type cells. This result is consistent with an earlier proposed model based on the assumption that polarization-type PID is caused by charge accumulation at K centers in SiN x layers. The findings described herein are crucially important for elucidating polarization-type PID and verifying the degradation model.

A case of posterior cerebral artery occlusion that developed into an artery of Percheron infarction.

The thalamus is predominantly supplied by multiple small vessels originating from the posterior communicating artery and the P1 and P2 segments of the posterior cerebral artery (PCA). The artery of Percheron (AOP) is a rare anatomical variant of arterial supply to the thalamus. This single thalamic perforating branch supplies the bilateral thalamus so that occlusion results in a characteristic cerebral infarction. Herein, we report a case of posterior cerebral artery occlusion that developed into an AOP infarction. A 74-year-old man, who had undergone coronary artery bypass grafting 5 days previously presented with sudden consciousness disorder and tetraplegia, and was admitted to our hospital. Magnetic resonance imaging (MRI) revealed a hyper-intense area in the bilateral paramedian thalamus on diffusion-weighted imaging and a deficit of the left PCA on MR angiography (MRA). The patient was diagnosed with cardiogenic cerebral embolism, and immediately underwent mechanical thrombectomy (MT), thereby complete recanalization was obtained. Post-procedural MRI showed no new lesions, and the left PCA could keep patency. His consciousness disorder and tetraplegia improved; however, cognitive impairment and vertical gaze palsy persisted as sequelae. To the best of our knowledge, such cases have not been previously reported. Additionally, in this case, we were able to identify an AOP on digital subtraction angiography, which was considered to be the responsible artery.

Asymmetric posterior reversible encephalopathy syndrome secondary to epilepsy occurring in the chronic phase of subarachnoid hemorrhage.

Background: Posterior reversible encephalopathy syndrome (PRES) is a rare clinical syndrome that refers to a disorder with reversible subcortical vasogenic brain edema involving the parieto-occipital lobe, temporal lobe, basal ganglia, and its surroundings. Radiologically, it is characterized by symmetrical lesions; however, atypical findings have sometimes been reported. Case Description: A 79-year-old woman experienced subarachnoid hemorrhage (SAH) a year and a half previously before this hospitalization. She presented with sudden-onset coma, dacryorrhea, and moderate right hemiparesis and was taken to our hospital. Computed tomography showed no apparent abnormal acute lesions. Electroencephalography confirmed periodic lateralized epileptiform discharges in the left hemisphere. First, based on the findings, she was diagnosed with nonconvulsive status epilepticus and started antiepileptic therapy. Six days after admission, however, multiple asymmetric lesions were confirmed on magnetic resonance imaging. Considering that findings subsequently improved, we finally diagnosed her with asymmetric PRES secondary to epilepsy occurring in the chronic phase of SAH. Aphasia and right hemispatial neglect persisted as sequelae and she was transferred to a rehabilitation hospital with a modified Rankin scale of 3. Conclusion: Excessive elevation of blood flow in the hemisphere is inferred to lead to blood-brain barrier collapse and subsequent asymmetric PRES.

Non-Destructive Measurement of Acetic Acid and Its Distribution in a Photovoltaic Module during Damp Heat Testing Using pH-Sensitive Fluorescent Dye Sensors.

An optical pH sensor that enables the non-destructive measurement of acetic acid and its distribution in a photovoltaic module during damp heat (DH) testing is reported. The sensor was fabricated by impregnating a solution of a pH-sensitive fluorescent dye into a fluororesin membrane filter, which was then dried. While conducting the DH test, fluorescence spectra from 20 pH sensors were periodically recorded and converted into pH values using a predetermined calibration curve. As a result, we succeeded in measuring changes in pH with a DH test time of up to 2000 h, and it was possible to obtain information on the pH distribution in the module. We also confirmed no change in pH in a module with a silicone encapsulant free from acetic acid, and revealed that the sensor that we developed does not respond to moisture and heat, but only to acetic acid.

High Protein Diet Feeding Aggravates Hyperaminoacidemia in Mice Deficient in Proglucagon-Derived Peptides.

(1) Background: Protein stimulates the secretion of glucagon (GCG), which can affect glucose metabolism. This study aimed to analyze the metabolic effect of a high-protein diet (HPD) in the presence or absence of proglucagon-derived peptides, including GCG and GLP-1. (2) Methods: The response to HPD feeding for 7 days was analyzed in mice deficient in proglucagon-derived peptides (GCGKO). (3) Results: In both control and GCGKO mice, food intake and body weight decreased with HPD and intestinal expression of Pepck increased. HPD also decreased plasma FGF21 levels, regardless of the presence of proglucagon-derived peptides. In control mice, HPD increased the hepatic expression of enzymes involved in amino acid metabolism without the elevation of plasma amino acid levels, except branched-chain amino acids. On the other hand, HPD-induced changes in the hepatic gene expression were attenuated in GCGKO mice, resulting in marked hyperaminoacidemia with lower blood glucose levels; the plasma concentration of glutamine exceeded that of glucose in HPD-fed GCGKO mice. (4) Conclusions: Increased plasma amino acid levels are a common feature in animal models with blocked GCG activity, and our results underscore that GCG plays essential roles in the homeostasis of amino acid metabolism in response to altered protein intake.

Risedronate Attenuates Podocyte Injury in Phosphate Transporter-Overexpressing Rats.

Osteoporosis patients with chronic kidney disease (CKD) are becoming common in our superaging society. Renal dysfunction causes phosphorus accumulation in the circulating plasma and leads to the development of CKD-mineral bone disorder (MBD). We have previously reported that type III Pi transporter-overexpressing transgenic (Pit-1 TG) rats manifest phosphate (Pi)-dependent podocyte injury. In the present study, we explored the effect of risedronate on Pi-induced podocyte injury in vivo. Pit-1 TG rats and wild-type rats at 5 weeks old were divided into a risedronate-treated group and an untreated group. We subcutaneously administered 5 µg/kg body weight of risedronate or saline twice a week during the experimental period. Risedronate did not alter serum creatinine levels at 5, 8, and 12 weeks of age. However, electron microscopy images showed that thickening of the glomerular basement membrane was improved in the risedronate treatment group. Furthermore, immunostaining for podocyte injury markers revealed that both desmin- and connexin43-positive areas were smaller in the risedronate-treated group than in the untreated group, suggesting that bisphosphonates could rescue Pi-induced podocyte injury. In conclusion, our findings suggest that risedronate could maintain glomerular barrier function by rescuing Pi-induced podocyte injury.

Low-carbohydrate diet by staple change attenuates postprandial GIP and CPR levels in type 2 diabetes patients.

AIMS: The aim of this study is to investigate the effects of a low-carbohydrate staple food (i.e., low-carbohydrate bread) on glucose and lipid metabolism and pancreatic and enteroendocrine hormone secretion in comparison with meals containing normal-carbohydrate bread, without consideration of the carbohydrate content of the side dishes. METHODS: T2DM patients (n = 41) were provided meals containing low-carbohydrate bread (LB) together with side dishes or normal-carbohydrate bread (NB) together with side dishes every other day as a breakfast. Blood glucose levels were evaluated by using a continuous glucose monitoring system; blood samples were collected before and 1 and 2 h after the breakfast. RESULTS: Postprandial blood glucose levels, plasma insulin, plasma glucose-dependent insulinotropic polypeptide (GIP) and plasma triglyceride were significantly lower and plasma glucagon levels were significantly higher in LB compared with those in NB. Plasma glucagon-like peptide-1 (GLP-1) levels did not differ in the LB and NB groups. CONCLUSIONS: These results indicate that changing only the carbohydrate content of the staple food has benefits on glucose and lipid metabolism in T2DM patients concomitant with the decrease of insulin and GIP secretion, which ameliorate body weight gain and insulin resistance.

Short-Term High-Starch, Low-Protein Diet Induces Reversible Increase in ß-cell Mass Independent of Body Weight Gain in Mice.

Long-term exposure to a high starch, low-protein diet (HSTD) induces body weight gain and hyperinsulinemia concomitantly with an increase in ß-cell mass (BCM) and pancreatic islets number in mice; however, the effect of short-term exposure to HSTD on BCM and islet number has not been elucidated. In the present study, we investigated changes in body weight, plasma insulin levels, BCM and islet number in mice fed HSTD for 5 weeks followed by normal chow (NC) for 2 weeks. BCM and islet number were increased in mice fed HSTD for 5 weeks compared with those in mice fed NC. On the other hand, mice fed HSTD for 5 weeks followed by NC for 2 weeks (SN) showed decreased BCM and insulin levels, compared to mice fed HSTD for 7 weeks, and no significant differences in these parameters were observed between SN and the control NC at 7 weeks. No significant difference in body weight was observed among HSTD, NC and SN fed groups. These results suggest that a high-starch diet induces an increase in BCM in a manner independent of body weight gain, and that 2 weeks of NC feeding is sufficient for the reversal of the morphological changes induced in islets by HSTD feeding.

Investigation of the characteristics of headache due to unruptured intracranial vertebral artery dissection.

BACKGROUND AND PURPOSE: It is sometimes difficult to diagnose intracranial vertebral artery dissection in patients with headache as the only symptom. Knowledge of the characteristics of the headache would facilitate the diagnosis. In this study, we aimed to clarify the characteristics of intracranial vertebral artery dissection-related headache using our original self-administered questionnaire. METHODS: Via the questionnaire, we ascertained headache characteristics and investigated whether they differed between two types of unruptured intracranial vertebral artery dissection, headache type and ischemic type, based on analysis of the responses. Then, we tried to validate the consistency of commonly used criteria for intracranial artery dissection by comparing them with our results. RESULTS: Thirty-seven patients were analyzed. Our results identified the following seven headache characteristics in patients with intracranial vertebral artery dissection: (i) occurring in the occipitonuchal region (89%); (ii) unilateral (81%); (iii) pulsatile (70%); (iv) of acute onset (70%); (v) severe (73%); (vi) without nausea or vomiting (73%); and (vii) with concomitant clinical symptoms unrelated to ischemia (81%). Comparison of headache characteristics between the two types of intracranial vertebral artery dissection headache showed that the pain was significantly more severe in headache type than ischemic type intracranial vertebral artery dissection ( p = 0.01). Concomitant clinical symptoms occurred significantly more often in ischemic type than headache type intracranial vertebral artery dissection ( p = 0.03). Our results generally satisfied the established headache diagnostic criteria. CONCLUSION: The pain characteristics of headache type and ischemic type intracranial vertebral artery dissection shown in our study may facilitate its diagnosis.

Which surgical procedure is effective for refractory chronic subdural hematoma? Analysis of our surgical procedures and literature review.

Refractory chronic subdural hematoma (CSDH) is rare but remains a difficulty for neurosurgeons, and no consensus on treatment procedures has been established. To discuss effective surgical procedures for refractory CSDH, we analyzed our surgical procedures and outcomes for refractory CSDH. We defined patients with refractory CSDH as those who presented with two or more recurrences. Fourteen patients with refractory CSDH were analyzed. Eight patients underwent burr-hole irrigation and closed-system drainage alone, four patients received embolization of the middle meningeal artery (MMA), and two patients with organized CSDH underwent large craniotomy with outer membranectomy as the third surgery. Two of the eight patients (25%) treated with burr-hole irrigation and drainage alone showed a third recurrence. No further recurrences were identified in patients treated with embolization of the MMA or craniotomy. However, statistical analysis showed no significant difference in cure rate between patients treated with burr-hole irrigation and drainage alone and patients treated with burr-hole irrigation and drainage with embolization of the MMA (P = .42). Similarly, no significant differences in cure rate were seen between patients treated with burr-hole irrigation and drainage alone and patients treated with craniotomy (P = .62). When selecting a surgical procedure, assessing whether the CSDH is organized is crucial. Embolization of the MMA may be considered as one of the optional treatments for refractory CSDH without organized hematoma. On the other hand, for refractory cases of organized CSDH, hematoma evacuation and outer membranectomy with large craniotomy or mini-craniotomy assisted by an endoscope may be suitable, as previous reports have recommended.

Clinical investigation of chronic subdural hematoma with impending brain herniation on arrival.

Chronic subdural hematoma (CSDH) with brain herniation signs is rarely seen in the emergent department. As such, there are few cumulative data to analyze such cases. In this study, we evaluated the clinical features, risk factors, and rates of completion with impending brain herniation on arrival in a cohort study. We analyzed 492 consecutive patients with CSDH between January 2010 and October 2015. First, we analyzed the clinical factors and compared them between patients with or without brain herniation signs on admission. Second, we compared clinical factors between patients with or without completion of brain herniation after operation among patients who had brain herniation signs on arrival. Eleven (2.2%) patients showed brain herniation signs on arrival, and six patients (1.2%) progressed to complete brain herniation. Patients with brain herniation signs on arrival were significantly older (P = 0.03) and more frequently hospitalized with a concomitant illness (P < 0.0001). Niveau formation (P = 0.0005) and acute-on CSDH (P = 0.0001) on computed tomography were also more frequently seen in patients with brain herniation signs. Multivariate logistic regression analysis showed that age older than 75 years (OR 2.16, P < 0.0001), niveau formation (OR 3.09, P < 0.0001), acute-on CSDH (OR 14, P < 0.0001), and admitted to another hospital (OR 52.6, P < 0.0001) were independent risk factors for having had brain herniation signs on arrival. On the other hand, having a history of head injury (P = 0.02) and disappearance of the ambient cistern (P = 0.0009) were significantly associated with completion of brain herniation. The prognosis was generally poor if the patient had presented with brain herniation signs on admission. Our results demonstrate that the diagnosis is often made late, despite hospitalization for a concomitant illness. When the elderly show mild disturbance of consciousness, physicians except neurosurgeons need to consider the possibility of CSDH regardless of a recent history of head injury.

Clinical Investigation of Refractory Chronic Subdural Hematoma: A Comparison of Clinical Factors Between Single and Repeated Recurrences.

BACKGROUND: Chronic subdural hematoma (CSDH) is sometimes refractory, and this is troublesome for neurosurgeons. Although many studies have reported risk factors or treatments in efforts to prevent recurrence, those have focused on single recurrence, and few cumulative data are available to analyze refractory CSDH. METHODS: We defined refractory CSDH as ≥2 recurrences, then analyzed and compared clinical factors between patients with single recurrence and those with refractory CSDH in a cohort study, to clarify whether patients with refractory CSDH experience different or more risk factors than patients with single recurrence, and whether burr-hole irrigation with closed-system drainage reduces refractory CSDH. RESULTS: Seventy-five patients had at least 1 recurrence, with single recurrence in 62 patients and ≥2 recurrences in 13 patients. In comparing clinical characteristics, patients with refractory CSDH were significantly younger (P = 0.04) and showed shorter interval to first recurrence (P < 0.001). Organized CSDH was also significantly associated with refractory CSDH (P = 0.02). Multivariate logistic regression analysis identified first recurrence interval <1 month (odds ratio, 6.66, P < 0.001) and age <71 years (odds ratio, 4.16, P < 0.001) as independent risk factors for refractory CSDH. On the other hand, burr-hole irrigation with closed-system drainage did not reduce refractory CSDH. CONCLUSIONS: When patients with risk factors for refractory CSDH experience recurrence, alternative surgical procedures may be considered as the second surgery, because burr-hole irrigation with closed-system drainage did not reduce refractory CSDH in our study.

Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis.

The dynamic assembly and disassembly of actin filaments is essential for the formation and transport of vesicles during endocytosis. In yeast, two types of actin structures, namely cortical patches and cytoplasmic cables, play a direct role in endocytosis, but how their interaction is regulated remains unclear. Here, we show that Srv2/CAP, an evolutionarily conserved actin regulator, is required for efficient endocytosis owing to its role in the formation of the actin patches that aid initial vesicle invagination and of the actin cables that these move along. Deletion of the SRV2 gene resulted in the appearance of aberrant fragmented actin cables that frequently moved past actin patches, the sites of endocytosis. We find that the C-terminal CARP domain of Srv2p is vitally important for the proper assembly of actin patches and cables; we also demonstrate that the N-terminal helical folded domain of Srv2 is required for its localization to actin patches, specifically to the ADP-actin rich region through an interaction with cofilin. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin cytoskeleton during clathrin-mediated endocytosis.

Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System.

The peptide-N (4)-(N-acetyl-ß-D-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins.

Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori.

DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn(2+) and Mn(2+). Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.

Expression, purification, and characterization of endo-ß-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system.

Endo-ß-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES.