Cepharanthine induces the proliferation of human dermal papilla cells and stimulates vascular endothelial growth factor expression through increased intracellular calcium mobilization and hypoxia-inducible factor activation.

BACKGROUND: Cepharanthine (CEP), a compound extracted from the vine Stephania cephalantha, is commonly prescribed to treat alopecia areata; however, the scientific evidence for its efficacy is limited. AIM: To investigate the effect of CEP and its structural analogues on human hair growth in vitro. METHODS: The effects of CEP and three of its structural analogues on the proliferation of human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs) were investigated. Their effects on vascular endothelial growth factor (VEGF) expression were also assessed by real-time PCR. Activation of pathways leading to VEGF expression, such as intracellular Ca2+ mobilization and hypoxia-inducible factor (HIF) expression, was also characterized. RESULTS: CEP and two of its structural analogues significantly stimulated the growth of hDPCs but not hORSCs. Moreover, CEP and all three structural analogues significantly induced the expression of VEGF in hDPCs. CEP increased the intracellular Ca2+ concentration in hDPCs. CEP also increased the expression of HIF-1α and HIF-2α and induced the expression of HIF-responsive genes in hDPCs, even under normoxia. CONCLUSIONS: These results suggest that CEP and its structural analogues have the potential to restore hair growth by promoting the proliferation of hDPCs and increasing their expression of VEGF.

Improvement of androgenetic alopecia with topical Sophora flavescens Aiton extract, and identification of the two active compounds in the extract that stimulate proliferation of human hair keratinocytes.

BACKGROUND: Androgenetic alopecia (AGA) is a hair loss disorder that commonly affects middle-aged men. To date, the properties of a number of natural or synthetic substances have been investigated for their ability to improve the condition. AIM: To evaluate the hair growth-promoting activities of an extract from the root of Sophora flavescens Aiton. METHODS: We used a human hair keratinocyte proliferation assay and ex vivo organ cultures of human hair follicle to examine the potential of the extract to stimulate hair growth via anagen elongation. We isolated the compounds promoting the growth of epithelial cells, and determined their chemical structures. A randomized, double-blinded, placebo-controlled clinical study for S. flavescens extract was carried out for 6 months with patients with AGA. RESULTS: The extract stimulated the proliferation of hair keratinocytes at a concentration of 0.1 ng/mL, while 100 ng/mL of the extract had a marked effect on hair shaft elongation in an organ culture of human hair follicle. Cell proliferation assay-directed fractionation led to the identification of two pterocarpan derivatives, L-maackiain and medicarpin, as active compounds that promote the proliferation of human hair keratinocytes. Studies in human subjects showed that improvement in the inspected alopecia scores in the lotion plus extract group were significant over a period of 6 months (P < 0.01). CONCLUSIONS: S. flavescens root extract is effective for the treatment of AGA. The isolated two pterocarpans might have important role in this effect.

Topical adenosine increases thick hair ratio in Japanese men with androgenetic alopecia.

OBJECTIVE: Hair thickness is more important than hair density in the appearance of baldness in male with androgenetic alopecia (AGA). Adenosine improves hair loss by stimulating hair growth and by thickening hair shafts in women. The objective of this study was to evaluate the hair growth efficacy and safety of topical adenosine in men with AGA. METHODS: A lotion containing either adenosine or niacinamide was administered to the scalps of 102 Japanese men twice daily for 6 months in a double-blind, randomized study. Efficacy was evaluated by dermatologists who assessed the quality of the hair and by calculating the percentages of vellus-like and thick hairs among the vertex hairs, as well as hair density. RESULTS: Adenosine was significantly (P < 0.05) superior to niacinamide in terms of global improvement of AGA, increase in the percentage of thick hairs (at least 60 µm) and self-assessment of hair thickness by the study participants. No causal adverse event due to the adenosine lotion was observed. CONCLUSION: These data indicate that adenosine increases thick hair ratio in Japanese men with AGA, and this compound is useful for the improvement of AGA.

Contribution of hair density and hair diameter to the appearance and progression of androgenetic alopecia in Japanese men.

BACKGROUND: Androgenetic alopecia (AGA) is the most common type of baldness in men. The balding process is associated with the gradual miniaturization of hair follicles and successive hair loss. However, the relative contributions of hair density and diameter to AGA are still unclear. OBJECTIVES: Hair density and hair diameter were investigated in Japanese men with or without AGA to elucidate the importance of these factors in the balding process. METHODS: Male Japanese subjects with or without AGA (n = 369) were included in this study. Hair appearance at the vertex was evaluated by comparison with a series of standard photographs. Hair density was measured using a phototrichogram-based videomicroscopy technique, and hair diameter was assessed by comparison with a series of calibrated threads on the phototrichogram image. RESULTS: All subjects with AGA were ≥ 25 years of age. The mean percentage of thick hairs (> 80 µm) in all subjects with AGA was significantly lower than that in subjects without AGA aged ≥ 25 years (P < 0·01), but the mean percentage of vellus hairs (< 40 µm) in subjects with AGA was significantly higher (P < 0·001). By contrast, the mean density of the hair in all patients with AGA did not significantly differ from the density of those without AGA aged ≥ 25 years. However, the mean density of the hair in subjects without AGA aged < 25 years was significantly higher than that of both subjects without AGA aged ≥ 25 years (P < 0·001) and all subjects with AGA. CONCLUSIONS: Hair loss in men with AGA results mainly from the miniaturization of hair follicles rather than the loss of hair (shedding), at least for individuals who are ≥ 25 years of age and present with AGA.

Reactive increase in gastric mucus secretion is an adaptive defense mechanism against low-dose aspirin-induced gastropathy.

BACKGROUND: Gastric mucus is considered to play an essential role in gastric mucosal defense mechanisms, especially when irritants are present in the stomach. AIM: To investigate the relationship between low-dose aspirin-induced gastropathy and gastric secretory function, especially gastric mucus secretion, in healthy volunteers. METHODS: Thirty male, asymptomatic, Helicobacter pylori pylori-negative healthy volunteers were asked to take 100 mg of enteric-coated aspirin (Bayaspirin) once a day for 10 days. Endoscopic examination was performed before and 3 and 10 days after drug administration. The extent of endoscopically assessed gastric mucosal injury was semi-quantitatively evaluated according to the modified Lanza score. The pentagastrin-stimulated gastric juice was collected for 10 min during the endoscopic examination and subjected to analysis for gastric acid (mEq/10 min) or mucus (mg hexose/10 min) output. RESULTS: Overall, the 10-day aspirin treatment significantly increased gastric mucus secretion from 0.8 (interquartile range 1.7) to 1.6 (1.6) mg hexose/10 min (P < 0.05), with a concomitant and significant decrease in the gastric acid/mucus ratio from 4.3 (5.2) to 2.9 (4.7) (P < 0.01). Subsequent analysis of two subgroups of volunteers categorized according to their endoscopic status ("severe gastropathy" vs. "modest gastropathy") revealed that changes in gastric secretory parameters occurred exclusively in those subjects without severe gastric injury; there was no alteration in these parameters in subjects with severe gastric injury. CONCLUSIONS: The results of this study suggest that the reactive increase in gastric mucus secretion is an adaptive defense mechanism against low-dose aspirin-induced gastropathy. In some individuals, such a response may be insufficient to prevent the development of severe mucosal injury and even ulcers and their complications.

Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice.

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

Bleeding tendency as a first symptom in children with congenital biliary dilatation.

AIM OF THE STUDY: Although a bleeding tendency as a first symptom is a critical condition in congenital biliary dilatation (CBD), the clinical details of this symptom remain unclear. We assessed this condition in children with CBD in this paper. MATERIALS AND METHODS: Sixty-five children with CBD were treated at our institute between 1983 and 2004. The children, initially presenting with bleeding manifestations such as intracranial hemorrhage and bloody stools, were defined as the bleeding group, and the remaining children with digestive symptoms such as abdominal pain and vomiting were defined as the digestive group. The clinical features were compared between these two groups. RESULTS: In 6 of the 65 cases, bleeding manifestations were noted (9.2 %). All six had cystic-type choledochal dilatation. The mean age of the bleeding group was significantly younger than that of the digestive group, and bleeding was more frequent, especially in infants less than 12 months of age. In a laboratory study, the bleeding group showed a more prolonged blood coagulation time than the digestive group did. Serum amylase and lipase levels in the bleeding group were almost normal, while those in the digestive group were significantly higher. The direct bilirubin level in the bleeding group was significantly higher than that in the digestive group. CONCLUSIONS: Disturbed blood coagulation due to vitamin K deficiency related to cholestasis results in a bleeding tendency in children with CBD. Therefore, pediatric surgeons should be aware of this rare but critical condition which can be prevented by rapid and precise treatment with vitamin K supplementation.

Gustatory sweating with special references to its mechanism.

A case of gustatory sweating associated with aneurysm in the right cervical region has been described. Gustatory sweating was observed not only on the right half of the face, but also on the right side of the neck and of the upper part of the frontal chest, on the right shoulder and the lateral side of the right arm. Thermoregulatory sweating was almost completely abolished in the involved area, but the responsiveness to intradermal acetylcholine of the sweat glands in the affected area was increased. Further, it was found that axon reflex sweating was elicited by intradermal injection of nicotine in the gustatory sweating area. Gustatory sweating in the present case can possibly be explained by assuming that an aberrant connection was formed by collateral sprouting from the efferent vagal nerve fibers into the preganglionic sympathetic sweat nerve fibers, very probably at about the level of the subclavian ansa.

A T7 promoter vector with a transcriptional terminator for stringent expression of foreign genes.

An expression vector, pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], was modified by replacing the trc promoter with the T7 phi 10 promoter and introducing a strong transcriptional terminator upstream from this promoter. This modification successfully suppressed background transcription and resulted in strict dependence upon induction.

Gene for a cyclophilin-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum.

A gene encoding a cyclophilin (CyP)-type peptidyl prolyl cis-trans isomerase (PPIase) was cloned from a halophilic archaeum, Halobacterium cutirubrum DSM 669, and sequenced. Although amino-acid residues common to CyPs were conserved, an insertion that showed no homology to other CyPs was found in its N-terminal region. Sequence analysis revealed that the amino-acid sequence of this CyP was 40-45% identical to those of eukaryotes and Bacillus subtilis with high cyclosporin A sensitivity, but 27% identical to those of cyclosporin A-insensitive PPIases of Escherichia coli. The gene was also expressed in E. coli. The activity of purified recombinant CyP-type PPIase was stimulated by the addition of KCl, and was suppressed by cyclosporin A.

FK506-binding protein-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum.

The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein.

Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products.

The method to internally label PCR products with multiple colored fluorescent dyes was developed and applied to multiple fluorescence-based PCR single-stranded conformational polymorphism (MF-PCR-SSCP) analysis. PCR-amplified fluorescent DNA fragments, which were internally labeled by adding fluorescent dUTPs ([F]dUTPs) to the PCR mixture, were heat-denatured and applied to a nondenaturing polyacrylamide gel (SSCP gel) set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by GENESCAN 672 software. In spite of differences in species and amount of integrated [F]dUTPs, the SSCP profiles were not significantly affected, even by the different labeling methods used-internal labeling or post-labeling at the 3' ends-in regard to the three different [F]dUTPs examined. However, the salt concentration of the solution containing the DNA samples affected the SSCP profiles. The internally labeled [F]dUTP-containing DNA fragments beyond 1000 bp in length were successfully digested with restriction endonucleases and subjected to SSCP analysis. MF-PCR-SSCP analysis with internal fluorescence labeling affords a simple and sensitive method to detect alterations in DNA sequences.

Effects of immunosuppressive peptidyl-prolyl cis-trans isomerase (PPIase) inhibitors, cyclosporin A, FK506, ascomycin and rapamycin, on hair growth initiation in mouse: immunosuppression is not required for new hair growth.

The effects of immunosuppressive peptidyl-prolyl cis-trans isomerase (PPIase) inhibitors, cyclosporin A, FK506, ascomycin and rapamycin, on hair growth initiation (anagen hair induction) in mouse were studied by topical application on the dorsal skin surface during the telogen phase of the hair cycle. Single applications of cyclosporin A and FK506 (10 to 100 nmol in 5 microliters of ethanol) induced new hair growth in 12 days within the restricted area where the compounds were applied. On the other hand, ascomycin and rapamycin did not initiate new anagen hairs even at higher doses (1 mumol in 5 to 10 microliters of ethanol). The effects of simultaneous application of the immunosuppressants were also tested by a single topical application. Ascomycin did not inhibit the anagen hair induction by cyclosporin A, but inhibited hair induction by FK506. Rapamycin inhibited new hair growth induced by cyclosporin A and FK506. These results suggest that the inhibition of PPIase is not required for the initiation of a new hair cycle in mice, and that anagen hair induction caused by cyclosporin A and FK506 is not a result of immunosuppression. The present results also indicate that a single application of an adequate quantity of cyclosporin A and FK506 is sufficient to initiate new hair growth.

Importance of monitoring the circulating blood volume in patients with cerebral vasospasm after subarachnoid hemorrhage.

We used the isotope dilution technique to monitor circulating blood volume (CBV) in three patients with ruptured cerebral aneurysms who developed pre- or postoperative ischemic symptoms that responded well to intravascular volume expansion therapy with blood transfusion and plasma expanders. In the first and second cases, predeterioration CBVs were obtained. Both of these patients showed hypovolemia and a decreased red blood cell volume at the time of neurological deterioration. A predeterioration CBV was not available for the third patient for comparison, but his red cell volume was also markedly decreased. Postrecovery CBVs were obtained in the second and third cases. Our data suggested that a depleted red blood cell volume was more responsible for neurological deterioration than was a lowered plasma volume. To prevent the occurrence of hypovolemia and anemia in aneurysm patients, we should monitor CBV not only at the time of neurological deterioration, but also at the time of admission and during the immediate postoperative period.

Prevention of cerebral ischemic symptoms in cerebral vasospasm with trapidil, an antagonist and selective synthesis inhibitor of thromboxane A2.

The results of our previous experimental and clinical studies led us to the hypothesis that, in the pathogenesis of cerebral vasospasm, subarachnoid focal acidosis resulting from anaerobic changes of subarachnoid clots may be a factor upsetting the balanced synthesis of both thromboxane A2 and prostaglandin I2 from prostaglandin endoperoxides on the inner surface of cerebral arteries. Thus, there is a higher concentration of thromboxane A2, a prostanoid that causes arterial contraction and platelet aggregation. We tested the administration of trapidil, an antagonist and selective synthesis inhibitor of thromboxane A2, in a series of 20 cases for the prevention of cerebral vasospasm and cerebral ischemia after aneurysmal rupture. Vasospasm was demonstrated by angiography in 9 of these cases, but only 2 of the 9 showed mild signs of cerebral ischemia. Of the 20 patients, 15 were discharged from the hospital as cured and 3 had a neurological deficit at discharge. Our findings suggest the significance in symptomatic vasospasm of thrombus formation by platelet aggregation and the effectiveness of trapidil as a preventive.

Changes in intracochlear and intracanalicular nerves after acoustic neurinoma excision confirmed by magnetic resonance imaging.

Postoperative magnetic resonance imaging findings related to the vestibulocochlear and facial nerves within the internal auditory canal were analyzed in acoustic neurinomas. T1- and proton-weighted magnetic resonance images showed that the vestibulocochlear nerves distal to the internal auditory meatus increased in signal intensity after surgical intervention. These nerves were conspicuously enhanced after intravenous administration of gadolinium diethylene-triamine-pentaacetic acid. The preserved facial nerves were also markedly enhanced postoperatively. As a possible cause of these findings, we suggest operative disruption of the blood-nerve barrier with ensuing nerve edema, although the operative procedures were carefully carried out using a surgical microscope. The clinical significance of traumatic disruption of the blood-nerve barrier and subsequent nerve edema are discussed from the standpoint of preservation of cochlear nerve function.

Vestibular nerve injury as a complication of microvascular decompression.

We report a case of hemifacial spasm in which hearing was well preserved after microvascular decompression, but the vestibular nerve was injured selectively. We review the pathophysiological mechanisms relevant to this complication in the light of results of animal experimental studies we have conducted. In addition, we discuss the clinical significance of this particular type of cranial nerve injury.

Intraoperative recordings of evoked extraocular muscle activities to monitor ocular motor nerve function.

During 22 operations in 18 patients, we stimulated the ocular motor nerves electrically, intracranially, and recorded compound muscle action potentials (CMAP) directly from the extraocular muscles with a ring electrode that we developed. Recording electrodes were applied in 52 instances to the superior rectus, medial rectus, superior oblique, or lateral rectus muscle and to the levator palpebrae superioris in 2 instances; CMAP were recorded successfully from 22 muscles. Evoked CMAP were not recorded in 2 instances because of problems with recording equipment; in the remaining 30 instances, no evoked CMAP were recorded because 1) the oculomotor or abducens nerve was not exposed during the operation; or 2) the recording electrode on the superior oblique muscle had not been properly placed to record trochlear nerve CMAP. Placement of this electrode is difficult. Ocular motor nerve function was analyzed preoperatively and postoperatively to evaluate the usefulness of this intraoperative electrophysiological monitoring method in preventing damage to ocular motor nerves. The results of this study showed that monitoring enables surgeons to locate precisely ocular motor nerves that would otherwise have been overlooked and thus possibly injured during surgery. Monitoring results also confirmed the surgeons' visual findings, thus helping the surgeons operate with greater confidence. Further, intraoperative monitoring provided us with some insights into the pathophysiology of ocular motor nerve dysfunction caused by skull base lesions; we documented electrophysiologically the occurrence of the slowing of conduction in the ocular motor nerves. We conclude that monitoring ocular motor nerve CMAP can reduce the incidence of surgical complications such as functional blindness due to inadvertent sectioning of one of these nerves and that it would be worthwhile to conduct studies of this technique in many more cases to validate our findings.

Changes of the auditory system after cerebellopontine angle manipulations.

The current tendency in acoustic neuroma surgery to attempt the preservation of hearing function and the problem of accidental hearing loss caused by microsurgical neurovascular decompression operations for hemifacial spasm or trigeminal neuralgia prompted us to study the exact surgical vulnerability of the auditory system. The surgical procedures for operation on the cerebellopontine angle of dogs were carried out according to the sequence of the posterior fossa transmeatal operation for acoustic neuroma. The operation was tentatively divided into three stages: (a) craniectomy and dural opening, (b) cerebellar retraction, and (c) identification of the cochlear nerve in the unroofed internal auditory canal (IAC). The postoperative behavior of the auditory system was evaluated electrocochleographically (EcochG) and histologically. Overzealous retraction of the cerebellar hemisphere caused transient disturbance of the EcochG pattern. Mechanical stretching of both the cochlear nerve and the internal auditory artery may cause a disturbance in the synchronized discharge of the cochlear neurons. Various manipulations at the porus acusticus internus or the IAC (such as pinching the nerve with forceps or electrocoagulation) produced thoroughly distorted EcochG patterns. From the histological findings, the main causative factor for these labyrinthine damages was considered to be vascular insufficiency. The current need for neurosurgical operations to preserve hearing is discussed in the light of these findings.