Hexyl aminolevulinate, 5-aminolevulinic acid nanoemulsion and methyl aminolevulinate in photodynamic therapy of non-aggressive basal cell carcinomas: A non-sponsored, randomized, prospective and double-blinded trial.

BACKGROUND: In the photodynamic therapy (PDT) of non-aggressive basal cell carcinomas (BCCs), 5-aminolevulinic acid nanoemulsion (BF-200ALA) has shown non-inferior efficacy when compared with methyl aminolevulinate (MAL), a widely used photosensitizer. Hexyl aminolevulinate (HAL) is an interesting alternative photosensitizer. To our knowledge, this is the first study using HAL-PDT in the treatment of BCCs. OBJECTIVES: To compare the histological clearance, tolerability (pain and post-treatment reaction) and cosmetic outcome of MAL, BF-200 ALA and low-concentration HAL in the PDT of non-aggressive BCCs. METHODS: Ninety-eight histologically verified non-aggressive BCCs met the inclusion criteria, and 54 patients with 95 lesions completed the study. The lesions were randomized to receive LED-PDT in two repeated treatments with MAL, BF-200 ALA or HAL. Efficacy was assessed both clinically and confirmed histologically at three months by blinded observers. Furthermore, cosmetic outcome, pain, post-treatment reactions fluorescence and photobleaching were evaluated. RESULTS: According to intention-to-treat analyses, the histologically confirmed lesion clearance was 93.8% (95% confidence interval [CI] = 79.9-98.3) for MAL, 90.9% (95% CI = 76.4-96.9) for BF-200 ALA and 87.9% (95% CI = 72.7-95.2) for HAL, with no differences between the arms (P = 0.84). There were no differences between the arms as regards pain, post-treatment reactions or cosmetic outcome. CONCLUSIONS: Photodynamic therapy with low-concentration HAL and BF-200 ALA has a similar efficacy, tolerability and cosmetic outcome compared to MAL. HAL is an interesting new option in dermatological PDT, since good efficacy is achieved with a low concentration.

A new method for characterizing hand dysfunction in cervical spondylotic myelopathy: a preliminary study.

STUDY DESIGN: A case-control investigation. OBJECTIVES: The objective of this study was to quantitatively study impaired ability to appropriately adjust pinch strength while holding a small object in patients with cervical spondylotic myelopathy (CSM). SETTING: Kochi Medical School Hospital, Japan. METHODS: The subjects consisted of 19 CSM patients who had frequent episodes of failing to grasp and hold small objects in their daily life (Group A), 13 CSM patients who did not experience such episodes (Group B) and 16 healthy subjects (Control Group). We continuously measured the dynamic internal pressure of a pneumatic rubber object called a blower pinched by the subject, following two different sets of instructions: (1) pinching with eyes open and with the minimal strength required to prevent dropping; and (2) maintaining a constant pinch strength at given levels with eyes closed. RESULTS: Compared with the other two groups, Group A subjects used a significantly (P<0.01) greater pinch strength to avoid dropping the blower held with eyes open and showed a significantly (P<0.01) greater deviation in pinch strength from the baseline values with eyes closed. These tendencies in Group A showed a significant correlation with the tactile perception threshold of the digits (P<0.01) but not with impairment of rapid repetitive movements of the digits that reflects spasticity. CONCLUSION: Our technique applied to CSM patients helps assess functional integrity primarily, if not exclusively, of the fasciculus cuneatus mediating the feedback signals from proprioceptive and cutaneous receptors in the digits, which are otherwise difficult to evaluate quantitatively.

Daylight photodynamic therapy for actinic keratoses: a randomized double-blinded nonsponsored prospective study comparing 5-aminolaevulinic acid nanoemulsion (BF-200) with methyl-5-aminolaevulinate.

BACKGROUND: Daylight-mediated photodynamic therapy (DL-PDT) using methyl-5-aminolaevulinate (MAL) is effective for thin, grade I, actinic keratoses (AK). There are no published studies of other photosensitizers used in DL-PDT. OBJECTIVES: To compare the efficacy and adverse effects of 5-aminolaevulinic acid nanoemulsion (BF-200 ALA) with MAL in DL-PDT of grade I-III AKs. METHODS: In 13 patients, 177 AKs were randomized symmetrically for a split-face prospective observer-blinded study and received either BF-200 ALA or MAL DL-PDT. Grade I AKs were treated once and grade II-III AKs twice with a 0·25-mm layer of photosensitizer precursors. Pain was assessed during and after the daylight exposure. Efficacy at 3 months was assessed clinically and histologically. RESULTS: BF-200 ALA cleared 71/84 (84·5%) and MAL 69/93 (74·2%) of the AKs (P = 0·099), all grades responding equally, but with new AKs appearing during follow-up (n = 4, BF-200 ALA; n = 8, MAL). In per patient half-face analysis BF-200 ALA showed significantly higher clearance rates for grade I AKs than did MAL (P = 0·027), but for thicker grades, clearance was equal (P = 0·564). BF-200 ALA and MAL treatments resulted in 61·5% and 38·5% complete histological clearance (P = 0·375), respectively. p53 expression decreased by 54·4% and 33·7%, respectively (P = 0·552). Both treatments were nearly painless with similar adverse reactions and no difference in patient preference. CONCLUSIONS: BF-200 ALA showed a trend towards improved efficacy results compared with MAL. Thicker lesions in both groups responded when treated repeatedly. Importantly, a thin 0·25-mm layer of the photosensitizer precursors was sufficient, which may lead to lower expense.

Maximum voluntary ventilation as a sensitive measure to monitor the ventilatory function in cervical spondylotic myelopathy.

STUDY DESIGN: A prospective clinical cohort study. OBJECTIVES: To test if maximum voluntary ventilation (MVV), which is currently underutilized in diseases, serves for assessing subclinical ventilatory impairment in cervical spondylotic myelopathy (CSM). SETTING: Kochi Medical School, Japan. METHODS: We studied ventilatory function in 49 CSM patients and 20 age- and sex-matched control patients with either lumbar stenosis or lower limb osteoarthritis. All patients underwent ventilatory function studies consisting of flow volume curves, vital capacity (VC) and the MVV in 12 s before and after surgery. Tetraparesis was assessed by the functional scale of the Japanese Orthopaedic Association (JOA). RESULTS: The CSM group had significantly smaller %forced VC , %peak expiratory flow rate (%PEFR) and %MVV than the control group preoperatively. In contrast to the control group, the CSM group showed a significant increase in %MVV from 74.9±18.7% preoperatively to 80.3±19.0% postoperatively (P<0.005), but not in any other ventilatory measures. This postoperative increase in %MVV significantly correlated with the JOA score (r=0.493; P<0.001). As a possible effect of diaphragmatic recovery, the %PEFR significantly increased postoperatively only in patients with the primary site of involvement at or rostral to C3-4. CONCLUSION: Of the various ventilatory measurements, MVV was most sensitive to changes in tetraparesis in CSM, presumably because MVV, unlike the other ventilatory measures, reflects the coordination in addition to the strength of respiratory muscles.

Nociceptive sensory innervation of the posterior cruciate ligament in osteoarthritic knees.

INTRODUCTION: Although the posterior cruciate ligament (PCL) is considered to contain not only proprioceptive but also nociceptive sensory fibers, there is a lack of information about nociceptive sensory innervation of the PCL. We hypothesized that the PCL has constant nociceptive sensory innervation, suggesting the possible source of osteoarthritic (OA) knee pain. MATERIALS AND METHODS: Innervation of the PCL was examined by immunohistochemistry with particular reference to nociceptive nerve fibers in OA knees. Sensory nerve fibers were semi-quantitatively counted in the PCL of OA knees, comparing with non-OA knees. Protein gene product 9.5 (PGP9.5) as a general neuronal marker and calcitonin gene related peptide (CGRP) as a marker for nociceptive neuron were used. RESULTS: The PCLs had constant CGRP-immunoreactive (IR) nerve fibers in both OA and non-OA knees. The difference of the CGRP-IR nerve density between groups did not reach a statistical significance (p = 0.062). For PGP9.5-IR nerve fibers, however, the PCLs in OA knees were statistically less innervated than non-OA knees (p = 0.0009). CONCLUSIONS: Our results showed that, in spite of a significant decrease in total innervation in OA knees, the PCLs have constant nociceptive sensory innervation. Although the relationship between the decrease in total innervations in the PCL and OA pathophysiology is still unclear, the PCL is the possible source of OA knee pain. Our results should be taken into account when examining the pain source of the OA knees and handling the PCL during total knee arthroplasty.

Development of a novel, nearly insoluble antiadhesive membrane.

BACKGROUND: Sodium hyaluronate/carboxymethylcellulose (HA/CMC) is difficult to use in a moist environment because of its susceptibility to moisture. METHODS: We developed the three-layered nDM-14R membrane. The surface layers are composed of 1-lactide, glycolide and e-caprolactone copolymers. HA/CMC and nDM-14R were used in all these studies. (1) The central region of 1 × 10 cm specimens (n = 5) was moistened for 0, 5, 10, 20, 30 or 60 s, after which the tensile strength was determined; (2) one side of specimens of 1 × 10 cm (n = 5) was moistened with agar gel for 5, 10, 15 or 30 s, after which the adhesion strength was determined, and (3) Rat cecum (n = 10) was scratched, 3 × 3 cm specimens were placed on the scratched area, and adhesions were evaluated on postoperative day 14. RESULTS AND CONCLUSION: (1) The tensile strength of nDM-14R after contact for 10-30 s was greater than that of HA/CMC. (2) The adhesive strength of HA/CMC after contact for 5-10 s was greater than that of nDM-14R. (3) Adhesion scores in treatment groups were significantly lower than in the control group. The results suggest that nDM-14R has the same antiadhesive effect and allows easier placement under moist conditions than HA/CMC.

Prevention of venous stasis in the lower limb by transcutaneous electrical nerve stimulation.

OBJECTIVES: This study aims to investigate the effects of thromboprophylactic transcutaneous electrical nerve stimulation (TpTENS) of the peroneal nerve on venous blood flow in the limbs of volunteers. TpTENS might be considered for use in preventing venous stasis during surgical treatment. METHODS: In 10 volunteers, peak venous velocity (PV) and flow volume (FV) in the popliteal vein were measured using duplex ultrasonography during calf-muscle stimulation. The effects of TpTENS of the peroneal nerve were compared with those of other mechanical methods, including electrical muscle stimulation, intermittent pneumatic compression, active ankle motion and calf squeeze, used to prevent venous stasis and achieve thromboprophylaxis. RESULTS: TpTENS had similar effects on popliteal vein blood flow in comparison with other established methods of thromboprophylaxis. The PV increased its basal flow by 3.9 times (p < 0.01) and FV by 2.7 times (p < 0.01), respectively, compared with baseline values. CONCLUSIONS: TpTENS is as effective as other electrical and mechanical methods of calf-muscle pump activation in achieving acceleration of venous flow in the lower limb.

Cross talk between hedgehog and epithelial-mesenchymal transition pathways in gastric pit cells and in diffuse-type gastric cancers.

We previously reported hedgehog (Hh) signal activation in the mucus-secreting pit cell of the stomach and in diffuse-type gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is known to be involved in tumour malignancy. However, little is known about whether and how both signallings cooperatively act in diffuse-type GC. By microarray and reverse transcription-PCR, we investigated the expression of those Hh and EMT signalling molecules in pit cells and in diffuse-type GCs. How both signallings act cooperatively in those cells was also investigated by the treatment of an Hh-signal inhibitor and siRNAs of Hh and EMT transcriptional key regulator genes on a mouse primary culture and on human GC cell lines. Pit cells and diffuse-type GCs co-expressed many Hh and EMT signalling genes. Mesenchymal-related genes (WNT5A, CDH2, PDGFRB, EDNRA, ROBO1, ROR2, and MEF2C) were found to be activated by an EMT regulator, SIP1/ZFHX1B/ZEB2, which was a target of a primary transcriptional regulator GLI1 in Hh signal. Furthermore, we identified two cancer-specific Hh targets, ELK1 and MSX2, which have an essential role in GC cell growth. These findings suggest that the gastric pit cell exhibits mesenchymal-like gene expression, and that diffuse-type GC maintains expression through the Hh-EMT pathway. Our proposed extensive Hh-EMT signal pathway has the potential to an understanding of diffuse-type GC and to the development of new drugs.

Eight calves cloned from somatic cells of a single adult.

Eight calves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). All calves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn calves.

The clinical significance of serum procalcitonin levels following direct hemoperfusion with polymyxin B-immobilized fiber column in septic patients with colorectal perforation.

BACKGROUND: The efficacy of direct hemoperfusion with polymyxin B-immobilized fiber columns (PMX) has already been demonstrated in clinical studies for the treatment of septic shock. However, serum procalcitonin levels following PMX remain unknown. METHODS: This prospective, multicenter, nonrandomized clinical study was performed at 12 institutions. Forty-five patients with severe sepsis or septic shock due to colorectal perforation underwent PMX. Patients' outcome as well as circulating levels of endotoxin, procalcitonin and IL-6 were monitored. RESULTS: Before surgery, procalcitonin level, but not endotoxin and IL-6 levels, was elevated according to patients' septic conditions. Procalcitonin was significantly and positively correlated with sequential organ failure assessment score. Circulating levels of procalcitonin peaked 24 h after PMX treatment. Change in serum procalcitonin level was significantly higher in nonsurvivors than survivors. Nine mortalities were observed within 28 days. The best predictor for 28-day mortality was procalcitonin >85.7 ng/ml at 24 h after PMX (area under the receiver operating characteristic curve: 0.808 +/- 0.105). CONCLUSIONS: Procalcitonin may be a good indicator of severity of sepsis secondary to colorectal perforation. Furthermore, procalcitonin level at 24 h after PMX appears to predict outcome after PMX. Therefore, procalcitonin may be a useful diagnostic marker to evaluate patients' condition in candidates for PMX treatment.

Effect of motion imagery to counter rest-induced suppression of F-wave as a measure of anterior horn cell excitability.

OBJECTIVE: To test if motor imagery prevents the rest-induced suppression of anterior horn cell excitability. METHODS: Ten healthy subjects underwent two separate experiments, each consisting of stimulating the median nerve 100 times and recording F-waves from abductor pollicis brevis (APB) in three consecutive sessions: (1) after muscle exercise to standardize the baseline, (2) after immobilization of APB for 3h and (3) after muscle exercise to check recovery. We instructed the subject to volitionally relax APB in experiment 1 (relaxation task), and to periodically simulate thumb abduction without actual movement in experiment 2 (imagery task). RESULTS: F-wave persistence and amplitude declined after relaxation task and recovered quickly after exercise, but changed little with imagery task. F-wave latencies showed no change when analyzed individually. The frequency distribution of collective F-waves recorded from all subjects remained the same after relaxation task, but showed a shift toward longer latencies after imagery task. CONCLUSIONS: Mental imagery without overt motor output suffices to counter the effect of sustained volitional muscle relaxation, which would, otherwise, cause a reversible reduction in anterior horn cell excitability. SIGNIFICANCE: This finding documents the importance of central drive for spinal excitability, which affects F-wave studies of a paretic muscle.

An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene.

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.

Nucleolar accumulation of poly (A)+ RNA in heat-shocked yeast cells: implication of nucleolar involvement in mRNA transport.

Transport of mRNA from the nucleus to the cytoplasm plays an important role in gene expression in eukaryotic cells. In wild-type Schizosaccharomyces pombe cells poly(A)+ RNA is uniformly distributed throughout the nucleoplasm and cytoplasm. However, we found that a severe heat shock blocks mRNA transport in S. pombe, resulting in the accumulation of bulk poly(A)+ RNA, as well as a specific intron-less transcript, in the nucleoli. Pretreatment of cells with a mild heat shock, which induces heat shock proteins, before a severe heat shock protects the mRNA transport machinery and allows mRNA transport to proceed unimpeded. In heat-shocked S. pombe cells, the nucleolar region condensed into a few compact structures. Interestingly, poly(A)+ RNA accumulated predominantly in the condensed nucleolar regions of the heat-shocked cells. These data suggest that the yeast nucleolus may play a role in mRNA transport in addition to its roles in rRNA synthesis and preribosome assembly.

Nucleolar accumulation of poly (A)+ RNA in heat-shocked yeast cells: implication of nucleolar involvement in mRNA transport.

Transport of mRNA from the nucleus to the cytoplasm plays an important role in gene expression in eukaryotic cells. In wild-type Schizosaccharomyces pombe cells poly(A)+ RNA is uniformly distributed throughout the nucleoplasm and cytoplasm. However, we found that a severe heat shock blocks mRNA transport in S. pombe, resulting in the accumulation of bulk poly(A)+ RNA, as well as a specific intron-less transcript, in the nucleoli. Pretreatment of cells with a mild heat shock, which induces heat shock proteins, before a severe heat shock protects the mRNA transport machinery and allows mRNA transport to proceed unimpeded. In heat-shocked S. pombe cells, the nucleolar region condensed into a few compact structures. Interestingly, poly(A)+ RNA accumulated predominantly in the condensed nucleolar regions of the heat-shocked cells. These data suggest that the yeast nucleolus may play a role in mRNA transport in addition to its roles in rRNA synthesis and preribosome assembly.

Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe.

Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.

A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase.

The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.

The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast.

The Schizosaccharomyces pombe dhp1(+) gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5'-->3' exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)(+) RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1(+) is required for proper chromosome segregation as well as for poly(A)(+) RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1(+). We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

Mapping regulatory networks in microbial cells.

Genome sequences are the blueprints of diverse life forms but they reveal little information about how cells make coherent responses to environmental changes. The combined use of gene fusions, gene chips, 2-D polyacrylamide gel electrophoresis, mass spectrometry and 'old-fashioned' microbial physiology will provide the means to reveal a cell's regulatory networks and how those networks are integrated.

Transport of steroid hormones facilitated by serum proteins.

The affinities with steroid hormones (alpha-estradiol, ethynylestradiol, progesterone, androsterone, dehydroisoandrosterone and testosterone) were observed for Cohn's fraction IV-1 and V (albumin). It was estimated from the comparison with the binding coefficient K (protein-bound form/free form of hormone) in a 3.5% (w/v) bovine serum albumin (BSA) solution that 40-80% of bound hormone in bovine serum is the BSA-bound form. It becomes clear in a liquid membrane system consisting of a hexane source phase (I), a water phase and a hexane receiving phase (II) that the transport flux of hormone is governed primarily by the partition coefficients between the water/hexane phases. In the case of a hormone with a lower partition coefficient, the uptake process from the hexane phase (I) to the water phase is a rate-determining step in the transport system and the serum proteins accelerate the transport of hormones, while with an increase in the partition coefficient the rate-determining step changes from the uptake step to the release step from the water phase to the hexane phase (II) and the hormone transport is decelerated owing to the significant decrease of free hormone concentration in the aqueous phase by the associated with serum proteins for the system having the restricted amount of hormone in the hexane source phase.