The usefulness of a free thinned deep inferior epigastric artery perforator flap and measurement of the vascular pedicle length: A thin flap with a long pedicle.

BACKGROUND: The thinned deep inferior epigastric perforator (DIEP) flap branching from the main trunk to the superolateral direction may be useful because of its long vascular pedicle. DIEP flap is used as an axial-pattern adipose flap. The vascular pedicle length of the thinned DIEP flap was investigated using originally developed software. The clinical application of the thinned DIEP flap was verified in a case series. METHODS: In 40 patients with enhanced computed tomography (CT) data, the vascular pedicle length of the longest thinned DIEP flap was simulated using the software. A free thinned DIEP flap was used in 10 clinical cases of facial or breast reconstruction. RESULTS: In all simulated cases, the vascular pedicle of the DIEP branching to the superolateral direction was the longest, and the vascular pedicle could be lengthened up to 34.8% by dissecting the vessels on the fascia as a vascular pedicle. In all the clinical cases, the reconstruction of a complex form defect or reconstruction requiring a long vascular pedicle could be achieved in one stage without any perioperative complications. The intraclass correlation coefficient between simulated pedicle length and dissected pedicle length was 0.99. CONCLUSION: Thinned DIEP flaps with long vascular pedicles could be elevated safely. Multiple adipose or muscle flaps could be combined without complications. The length of the winding vascular pedicle could be measured using imaging data using the software first developed in the present study. This software would be useful in the planning of a thinned DIEP flap and other free flaps.

Comparison of Vectra three-dimensional stereophotogrammetry measurement and tape measurement in the evaluation of perioperative volume change of the lower abdomen in association with lymphatic microsurgery.

BACKGROUND: Although microsurgical treatment for lower extremity lymphedema (LEL) can improve lower abdominal morphology, methods to evaluate the volume change of the lower abdomen have yet to be established. This study aimed to determine the accuracy and reproducibility of three-dimensional stereophotogrammetry (3DSM) in measuring the volume change in the lower abdomen. METHODS: The perioperative volume changes in the lower abdomen were estimated using tape measurement (TM) and 3DSM in 26 patients with LEL. Thirteen patients with suprapubic lymphedema underwent abdominoplasty simultaneously. Each of them underwent multiple lymphaticovenular anastomoses (LVAs), and five of them underwent vascularized lymph node transfer, simultaneously. Thirteen patients with pelvic lymphatic fluid underwent multiple LVAs. Two patients underwent this surgery twice. When assessed on the Internal Society of Lymphology scale, eight patients were Stage I, 10 patients were Stage II, four patients were late Stage II, and four patients were Stage III. The difference between the two measurement methods and reproducibility of each method were analyzed. RESULTS: During a mean follow-up period of 6 months, all patients had no postoperative complications and their chief complaint improved. The calculated reduction volume between TM and 3DSM showed a high correlation (p < .0001, r = .84). The reduction volume based on TM was significantly larger than 3DSM (991.1 ± 460.3 ml vs. 862.3 ± 333.5 ml, p = .02). The interrater ICC was 0.94 and 0.98 based on TM and 3DSM, respectively. CONCLUSION: 3DSM may be a useful method for assessment of the lower abdominal morphology due to its high accuracy and reproducibility.

Noninvasive, objective evaluation of lower extremity lymphedema severity using shear wave elastography: A preliminary study.

BACKGROUND: Increased skin and subcutaneous tissue stiffness in patients with early-stage lymphedema has been reported. The purpose of this study was to examine the use of shear wave elastography (SWE) for evaluating lower extremity lymphedema (LEL). METHODS: For 10 lower extremities of normal controls and 72 limbs of patients with gynecological cancer whose lymphatic function was categorized into six stages based on the range of dermal backflow (DBF) observed in indocyanine green (ICG) lymphography, SWE was performed and shear wave velocity (SWV) of the dermis and three layers of subcutaneous tissue at the thigh and calf were recorded. Twenty-five patients underwent thigh tissue histological and dermal thickness examinations. RESULTS: The strongest correlation between the ICG DBF stage and SWV during SWE was observed on the dermal layer of the thigh (p < 0.01, R = 0.67). There was a significant correlation between the dermal thickness of the thigh and the ICG DBF stage (p < 0.01, R = 0.87) and also between the dermal thickness of the thigh and SWV (p < 0.01, R = 0.73). CONCLUSION: Noninvasive, objective evaluation of LEL severity using SWE was well correlated with lymphatic function as determined by ICG lymphography. The DBF changes in the dermis of the thigh best reflected the changes in lymphatic function. Dermal thickness variations may partially account for differences in SWV.

Where does subcutaneous lymph from the chest wall flow into after mastectomy?

BACKGROUND: Changes of the lymph flow from the chest wall after mastectomy and sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (Ax) has yet to be understood. This study aimed to investigate the effect of axillary surgery on lymphatic flow from the chest wall in patients who have undergone mastectomy, including those have undergone breast reconstruction and vascularized lymph node transfer (VLNT). METHODS: Following mastectomy in 100 breasts, the directions of lymph flow from the chest wall was compared between the SLNB omission, SLNB, Ax, and Ax followed by VLNT groups using indocyanine green (ICG) lymphography in cross-sectional study. Lymph flow on the deep epigastric artery perforator (DIEP) flap was also investigated. RESULTS: Lymph flow directing to the ipsilateral axilla was observed more frequently after SLNB than Ax (48% vs. 12.5%; p = 0.005); however, no significant difference was observed in the frequency of contralateral axillary route adoption between them (8% vs. 15%; p = 0.65). In the VLNT group, lymph flow to the ipsilateral axilla was not observed at a significantly higher frequency than in the Ax group (12.5% vs. 12.5%, p = 1.00). On the transferred DIEP flap, the lymph flowed anterograde or retrograde parallel to the anatomic course of the lymphatic vessels. CONCLUSION: To visualize the direction of lymph flow of the chest following mastectomy, ICG lymphography may be useful to discern the direction in which malignant neoplasms, including lymphoma, are transported and to plan for lymph flow restoration.

Internal Mammary Vein Valves: A Histological Study.

It is widely accepted that the internal mammary vein (IMV) is valveless. However, few anatomical studies are available on the presence or absence of IMV valves. To test the hypothesis that the IMV is valveless, we performed microscopic histological examination of the IMV. IMV samples were collected from 10 human fresh frozen cadavers. For a control, the small saphenous vein (SSV) was obtained. Histological stains were performed. Microscopic examination showed that a venous valve was found in 8 of 20 IMVs. The structure of the valve leaflet consisted of two parts. There was a "thick part" located near the wall of the vein that consisted of smooth muscle cells and fibers. There was also a "thin part" located near the center of the venous lumen that lacked smooth muscle cells. The size of the thick part of the IMV valve was smaller than the SSV valve, whereas there was no difference in the size of the thin part between the IMV and SSV. IMV valves exist. Our results that an IMV valve was present in less than half of IMVs and there was a small-sized valve leaflet suggest that the IMV valve may be rudimentary.

Prevention of venous reflux with full utilization of venoplasty in lymphaticovenular anastomosis.

BACKGROUND: Intraoperative retrograde blood flow from the vein to the lymphatic vessels in lymphaticovenular anastomosis (LVA) for lower extremity lymphedema (LEL) leads to poor results. This study aimed to establish a treatment strategy to control venous reflux in LVA. METHODS: A unified strategy to prevent venous reflux was used in 95 limbs (study group). Dilated perforating veins were ligated, and LVA at the small branch of the ligated vein was considered. External valvuloplasty in the small vein was performed to eliminate venous reflux pre- and post-LVA. A Y-shaped venoplasty for the relatively large vein was considered in cases without adequate-sized vein stump with a functional valve. The results were compared with the 34 limbs undergoing conventional multiple LVAs (control group). RESULTS: Intraoperative venous reflux and postoperative ecchymosis significantly decreased in the study group (0/462 anastomosis vs. 15/148 anastomosis, p < 0.0001 and 0/81 patients vs. 3/25 patients, p = 0.01, respectively). The average frequency of cellulitis during a year following LVA was significantly smaller in the study group than in the control group (0.05 ±â€¯0.03 vs 0.20 ±â€¯0.06, p = 0.04).The amount of improvement in the LEL index a year after LVA was significantly larger in the study group than in the control group (22.2 ±â€¯9.6 vs. 18.3 ±â€¯9.8, p = 0.04). CONCLUSION: Using the new strategy developed in this study, venous reflux could be completely prevented, and stable clinical results were obtained in patients with LEL. Prevention of venous reflux with full utilization of venoplasty might improve the LVA result.

The beneficial effect of traditional Japanese herbal (Kampo) medicine, Hochu-ekki-to (Bu-Zhong-Yi-Qi-Tang), for patients with chronic wounds refractory to conventional therapies: A prospective, randomized trial.

Hochu-ekki-to (HET) is a traditional Japanese herbal (Kampo) medicine for the treatment of severe weakness, loss of appetite, and indigestion in elderly patients and for the prevention of opportunistic infections. The impact of HET on patients with chronic wounds refractory to conventional therapies was investigated in a prospective, randomized trial, including 18 patients divided into medication (7.5 g oral HET per day, n = 9) and control (n = 9) groups. Wound healing during the 12-week study period was scored based on depth, exudate, size, inflammation/infection, granulation tissue, necrotic tissue, and pocket size. At 12 weeks, wound healing progressed in all nine patients in the medication group, whereas wound healing progressed in only three patients in the control group (significant difference, p < 0.01; relative risk: 3.00). In the medication group, the total score decreased significantly at 8 weeks and later. To the best of our knowledge, this study was the first to show that HET promoted the healing of chronic wounds resistant to conventional treatments. HET may be a choice as an adjunctive therapy for chronic wounds, particularly for patients with malnutrition. This trial was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN000031620).

Disseminated zoster in an adult patient with extensive burns: a case report.

BACKGROUND: Shingles (localized zoster) and disseminated zoster are caused by the reactivation of latent varicella zoster virus (VZV). Reactivation of VZV is related to impaired cell-mediated immunity. Extensive burns affecting a patient result in burn-related immunosuppression and cytokine storm. Despite immunosuppression in burn patients, the reactivation of VZV is extremely rare, whereas eczema herpeticum, caused by reactivation of latent herpes simplex virus (HSV), is common. We have found only 1 published case of VZV reactivation during burn treatment in the literature. CASE PRESENTATION: A 51-year-old man was burned in a fire, which affected 60% of his total body surface area (TBSA), and also received inhalation injury (day 0). Despite fluid resuscitation, he showed persistent renal failure. Continuous hemodialysis and filtration (CHDF) combined with polymyxin B-immobilized fiber column direct hemoperfusion (PMX-DHP) therapy was used for cytokine modulation. Autologous and allogeneic skin grafting was performed. On day 15, multiple-drug-resistant Pseudomonas aeruginosa (MDRP) was detected from a blood specimen, and the patient developed multiple organ failure (MOF). On day 31, compact aggregations of small vesicles appeared on the intact skin of his left knee and left buttock. The vesicles were located within the 4th lumbar (L4) spinal dermatome. From day 32 to day 34, similar new vesicles arose on his intact skin and epithelializing skin-graft donor sites. We diagnosed disseminated zoster, based on the patient's age, the characteristic occurrence of the initial vesicles within a limited area of intact skin in the left L4 dermatome, and a positive Tzank smear. Serologic testing on day 36 showed a high level of anti-VZV immunoglobulin (Ig)G with low levels of anti-VZV IgM, anti-HSV IgG, and anti-HSV IgM. The patient was isolated in a negative-pressure room to avoid air-borne spread of VZV. On day 52, the patient died. CONCLUSIONS: To the best of our knowledge, our patient is the second case of reactivation of VZV during burn treatment. It is unclear why reactivation of VZV is rare in patients with burn-related immunosuppression, whereas HSV reactivation is common. Cytokine modulation throughout the treatment period using CHDF combined with PMX-DHP might have been related to the rare reactivation of VZV in our patient. Our case provides an additional information on the relationship between the immune status of a patient with extensive burns and reactivation of latent VZV or HSV.

Intraoperative Detection of Efferent Lymphatic Vessels Emerging from Lymph Node during Lymphatic Microsurgery.

BACKGROUND: Although the usefulness of efferent lymphaticovenular anastomosis (ELVA) in lymphatic microsurgery has been reported, the optimal method to distinguish efferent from afferent lymphatics is not yet established. We propose a novel technique to detect efferent lymphatics appropriate for anastomosis. METHODS: In total, 62 groin lymph nodes (LNs) of 46 limbs were divided into four groups based on the findings of indocyanine green lymphography: n = 15 in normal, 15 in dermal backflow stage 0, 18 in stage I, and 14 in stage II groups. The target LN and afferent lymphatic connecting it were preoperatively detected using ultrasonography. Intraoperatively, 0.05 mL patent blue dye was slowly manually injected from the afferent lymphatic; the lymphatic(s) subsequently stained was diagnosed as the efferent lymphatic(s) emerging from the node. The success rates of efferent lymphatic detection, sizes of LN, and diameter of efferent lymphatics were compared among the groups. RESULTS: Both LN size and diameter of efferent lymphatic were significantly larger in the stage 0 and I groups than the other groups (p < 0.01). Efferent lymphatic could be stained in 13, 13, 18, and 9 LNs in the normal and stage 0, I, and II groups, respectively. A significant difference was observed between the stage I and II groups regarding the success rate of efferent lymphatic detection (p = 0.04). CONCLUSION: Efferent lymphatics could be detected using patent blue dye in 85.5% of the cases. The patients in early-stage lymphostasis might be the most appropriate candidates for ELVA.

Total Nasal Reconstruction with a Nonlaminated Vascularized Free Temporal Fascia as the Lining.

Various methods to generate the lining for a full-thickness nasal reconstruction have been reported. We used bilateral septal mucoperichondrial flaps, the distal portion of an expanded median forehead flap, and a nonlaminated vascularized free temporal fascia flap as a lining during total nasal reconstruction of a total full-thickness nasal defect in a 45-year-old woman with a nasal squamous cell carcinoma. In the first step of the two-stage surgery, a tissue expander was inserted into the forehead simultaneously with tumor resection. In the second step, the expanded median forehead flap, cartilage graft, bilateral septal mucoperichondrial flaps, and short pedicle vascularized free temporal fascia transfer were performed. Total nasal reconstruction could be completed without any skin graft or skin paddle of the free flap. Epithelialization of the mucosa on the transferred vascularized free temporal fascia without contracture deformity of the nasal cavity was confirmed by endoscopic examination after 8 years of follow-up. In the surgical procedure described, the facial skin, including the lining of the nostril rim, and the mucous membrane of the nasal cavity were reconstructed using facial skin and mucous membrane without long-term contracture, respectively.

Intraoperative objective evaluation of venous congestion in deep epigastric artery perforator flap breast reconstruction: A pilot study.

BACKGROUND: In a deep epigastric artery perforator (DIEP) flap breast reconstruction, the necessity of additional anastomosis of the superficial inferior epigastric vein (SIEV) should be determined intraoperatively. The purpose of this pilot study is to propose a method to detect venous congestion intraoperatively using the blood glucose measurement index (BGMI). METHODS: In 70 DIEP flaps of 67 patients for breast reconstruction, the ratio of blood glucose content in the flap to systemic blood glucose (BGMI) was measured immediately after wound closure. Eight flaps in which BGMI was less than 0.8 were categorized into the low BGMI group, and additional venous anastomosis using SIEV was conducted. The other 62 flaps were categorized into the normal BGMI group. Perioperative objective color difference of the flap calculated using the L*a*b* color coordinate scale was recorded simultaneously. Correlation between BGMI and color difference was analyzed. RESULTS: In all flaps in the low BGMI group, SIEV diameter was increased; after additional anastomosis, the BGMI significantly improved (from 0.71 ± 0.05 to 0.94 ± 0.05, P < .01). There was a significant correlation between BGMI and color difference (P = .04). The determination coefficient was 0.265. When a BGMI of less than 0.8 was assumed to be a true positive, the area under the curve of color difference in the receiver operating characteristic curve was 0.82. CONCLUSION: BGMI immediately after wound closure may be useful to detect initial signs of venous congestion. Intraoperative objective color difference also reflects venous congestion; however, it is not highly accurate.

Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain.

We continuously monitored bioluminescence from a wild-type reporter strain of Escherichia coli (lacp::luc+/WT), which carries the promoter of the lac operon (lacp) fused with the firefly luciferase gene (luc+). This strain showed a bioluminescence burst when shifted into the stationary growth phase. Bioluminescence profiles of other wild-type reporter strains (rpsPp::luc+ and argAp::luc+) and gene-deletion reporter strains (lacp::luc+/crp- and lacp::luc+/lacI-) indicate that transcriptional regulation is not responsible for generation of the burst. Consistently, changes in the luciferase protein levels did not recapitulate the profile of the burst. On the other hand, dissolved oxygen levels increased over the period across the burst, suggesting that the burst is, at least partially, caused by an increase in intracellular oxygen levels. We discuss limits of the firefly luciferase when used as a reporter for gene expression and its potential utility for monitoring metabolic changes in cells.

Near-UV radiation acts as a beneficial factor for physiological responses in cucumber plants.

Effects of near-UV radiation on the growth and physiological activity of cucumber plants were investigated morphologically, physiologically and biochemically using 3-week-old seedlings grown under polyvinyl chloride films featuring transmission either above 290 nm or above 400 nm in growth chambers. The hypocotyl length and leaf area of cucumber seedlings were reduced but the thickness of leaves was enhanced by near-UV radiation, due to increased upper/lower epidermis thickness, palisade parenchyma thickness and volume of palisade parenchyma cells. Photosynthetic and respiratory activities were also promoted by near-UV radiation, associated with general enhancement of physiological/biochemical responses. Particularly, metabolic activities in the photosynthetic system of chloroplasts and the respiratory system of mitochondria were analyzed under the conditions of visible light with and without near-UV radiation. For example, the activities of NAD(P)-dependent enzymes such as glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in chloroplasts and isocitrate dehydrogenase (ICDH) in mitochondria were elevated, along with levels of pyridine nucleotides (nicotinamide coenzymes) [NAD(H) and NADP(H)] and activity of NAD kinase (NADP forming enzyme). Taken together, these data suggest that promotion of cucumber plant growth by near-UV radiation involves activation of carbon and nitrogen metabolism in plants. The findings of this research showed that near-UV radiation reaching the Earth's surface is a beneficial factor for plant growth.

Bottle gourd rootstock-grafting affects nitrogen metabolism in NaCl-stressed watermelon leaves and enhances short-term salt tolerance.

The plant growth, nitrogen absorption, and assimilation in watermelon (Citrullus lanatus [Thunb.] Mansf.) were investigated in self-grafted and grafted seedlings using the salt-tolerant bottle gourd rootstock Chaofeng Kangshengwang (Lagenaria siceraria Standl.) exposed to 100mM NaCl for 3d. The biomass and NO3(-) uptake rate were significantly increased by rootstock while these values were remarkably decreased by salt stress. However, compared with self-grafted plants, rootstock-grafted plants showed higher salt tolerance with higher biomass and NO3(-) uptake rate under salt stress. Salinity induced strong accumulation of nitrate, ammonium and protein contents and a significant decrease of nitrogen content and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) in leaves of self-grafted seedlings. In contrast, salt stress caused a remarkable decrease in nitrate content and the activities of GS and GOGAT, and a significant increase of ammonium, protein, and nitrogen contents and NR activity, in leaves of rootstock-grafted seedlings. Compared with that of self-grafted seedlings, the ammonium content in leaves of rootstock-grafted seedlings was much lower under salt stress. Glutamate dehydrogenase (GDH) activity was notably enhanced in leaves of rootstock-grafted seedlings, whereas it was significantly inhibited in leaves of self-grafted seedlings, under salinity stress. Three GDH isozymes were isolated by native gel electrophoresis and their expressions were greatly enhanced in leaves of rootstock-grafted seedlings than those of self-grafted seedlings under both normal and salt-stress conditions. These results indicated that the salt tolerance of rootstock-grafted seedlings might (be enhanced) owing to the higher nitrogen absorption and the higher activities of enzymes for nitrogen assimilation induced by the rootstock. Furthermore, the detoxification of ammonium by GDH when the GS/GOGAT pathway was inhibited under salt stress might play an important role in the release of salt stress in rootstock-grafted seedlings.

Effects of 24-epibrassinolide on the photosynthetic characteristics, antioxidant system, and chloroplast ultrastructure in Cucumis sativus L. under Ca(NO(3))(2) stress.

The effects of 0.1 µM 24-epibrassinolide (EBL) on plant growth (plant height, leaf area, fresh weight, and dry weight), chlorophyll content, photosynthetic characteristics, antioxidant enzymes, and chloroplast ultrastructure were investigated using cucumber seedlings (Cucumis sativus L. cv. Jinyou No. 4) with 80 mM Ca(NO(3))(2) to induce stress. The presence of Ca(NO(3))(2) caused significant reductions in net photosynthetic rate (P(N)), stomatal conductance (Gs), intercellular CO(2) concentration (Ci), and transpiration rate (Tr) of leaves. In addition, Ca(NO(3))(2) markedly reduced the chlorophyll content and inhibited photochemical activity, including the actual photochemical efficiency (ΦPSII). In contrast, EBL increased the chlorophyll content, especially chlorophyll b, and minimized the harmful effects on photosynthesis caused by the Ca(NO(3))(2). The application of EBL to the plants subjected to Ca(NO(3))(2)-enhanced photochemical activity. EBL protected the photosynthetic membrane system from oxidative damage due to up-regulating the capacity of the antioxidant systems. Microscopic analyses revealed that Ca(NO(3))(2) affected the structure of the photosynthetic apparatus and membrane system and induced damage of granal thylakoid layers, while EBL recovered the typical shape of chloroplasts and promoted the formation of grana. Taken together, EBL compensated for damage/losses by Ca(NO(3))(2) due to the regulation of photosynthetic characteristics and the antioxidant system.

Proteomic study participating the enhancement of growth and salt tolerance of bottle gourd rootstock-grafted watermelon seedlings.

An insertion grafting technique to do research on salt tolerance was applied using watermelon (Citrullus lanatus [Thunb.] Mansf. cv. Xiuli) as a scion and bottle gourd (Lagenaria siceraria Standl. cv. Chaofeng Kangshengwang) as a rootstock. Rootstock-grafting significantly relieved the inhibition of growth and photosynthesis induced by salt stress in watermelon plants. Proteomic analysis revealed 40 different expressed proteins in response to rootstock-grafting and/or salt stress. These proteins were involved in Calvin cycle, amino acids biosynthesis, carbohydrate and energy metabolism, ROS defense, hormonal biosynthesis and signal transduction. Most of these proteins were up-regulated by rootstock-grafting and/or susceptible to salt stress. The enhancement of the metabolic activities of Calvin cycle, biosynthesis of amino acids, carotenoids and peroxisomes, glycolytic pathway and tricarboxylic acid cycle will probably contribute to intensify the biomass and photosynthetic capacity in rootstock-grafted seedlings under condition without salt. The accumulation of key enzymes included in these biological processes described above seems to play an important role in the enhancement of salt tolerance of rootstock-grafted seedlings. Furthermore, leucine-rich repeat transmembrane protein kinase and phospholipase may be involved in transmitting the internal and external stimuli induced by grafting and/or salt stress.

Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus.

Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint.

Proteomic analysis of cucumber seedling roots subjected to salt stress.

To understand metabolic modifications in plants under salt stress, the physiological and biochemical responses of cucumber (Cucumis sativus L. cv. Jinchun No. 2) seedlings to salt stress was investigated. The dry weight and fresh weight of cucumber seedling roots were significantly reduced by treatment with NaCl; Na(+) and Cl(-) were increased, while K(+) and K(+)/Na(+) ratio were decreased. To identify components of salt stress signaling, we compared the high resolution two-dimensional gel electrophoresis (2-DE) protein profiles of control and NaCl-treated roots, and the intensity of 34 protein spots varied. Of these spots, the identities of 29 (21 up-regulated and 8 down-regulated protein spots induced after salt stress) were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS). The majority of the proteins had functions related to metabolism, energy and transport, and are involved in regulating reactions and defending against stress. A semi-quantitative reverse transcriptional-polymerase chain reaction (PCR) approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 10 candidate proteins. Of these proteins, 8 patterns of induced transcript accumulation were consistent with those of induced protein accumulation. It is therefore likely that the response of the plant's proteome to NaCl stress is complex, and that the identified proteins may play an important role in regulating adaptation activities following exposure to NaCl stress in order to facilitate ion homeostasis.

Differences in the P1' substrate specificities of pepsin A and chymosin.

Porcine pepsin A and bovine chymosin are typical models of aspartic proteinases. The hydrolytic specificities of these proteinases, along with those of human pepsin A and monkey chymosin, were investigated with 29 peptide substrates that included various P1' variants of seven parent peptides. From these peptides, AFPLEF downward arrow FREL was preferred by pepsin A and chymosin, while its P1' variant, AFPLEF downward arrow EREL was preferred by bovine chymosin. Porcine and human pepsin A showed similar hydrolytic specificities, strongly preferring a hydrophobic/aromatic residue at P1' of any type of peptide. This specificity is well explained by the very hydrophobic nature of the S1' subsite that consists of Tyr(189), Ile(213), Ile(300), Met(289), Val/Leu(291) and Leu(298). The first three residues are well conserved in pepsin family enzymes. Although bovine and monkey chymosin showed similar P1' specificity, bovine chymosin preferred peptides having Glu at P1', while monkey chymosin preferred peptides having Lys at P1'. The dual characteristics of chymosin are due to the occurrence of polar/charged residues in the S1' subsite, such as Glu/Asp(289), Gln(298) and Lys/Gln(299), which are different from the S1' subsite of pepsin A. Molecular models suggest that Glu in position 289 of bovine chymosin and Asp in position 289 of monkey chymosin are responsible for the difference in P1' specificities between the chymosins.

Effects of exogenous putrescine on gas-exchange characteristics and chlorophyll fluorescence of NaCl-stressed cucumber seedlings.

The effects of 10 mM putrescine (Put) treated by spraying on leaves on growth, chlorophyll content, photosynthetic gas-exchange characteristics, and chlorophyll fluorescence were investigated by growing cucumber plants (Cucumis sativus L. cv. ChangChun mici) using hydroponics with or without 65 mM NaCl as a salt stress. Salt stress caused the reduction of growth such as leaf area, root volume, plant height, and fresh and dry weights. Furthermore, net photosynthesis rate (P(n)), stomatal conductance (g(s)), intercellular CO(2) concentration (C(i)), and transpiration rate (T(r)) were also reduced by NaCl, but water use efficiency (WUE; P(n)/T(r)) showed a tendency to be enhanced rather than reduced by NaCl. However, Put alleviated the reduction of P (n) by NaCl, and showed a further reduction of C (i) by NaCl. The reduction of g(s) and T(r) by NaCl was not alleviated at all. The enhancement of WUE by NaCl was shown to have no alleviation at day 1 after starting the treatment, but after that, the enhancement was gradually reduced till the control level. Maximum quantum efficiency of PSII (F(v)/F(m)) showed no effects by any conditions based on the combination of NaCl and Put, and in addition, kept constant values in plants grown in each nutrient solution during this experimental period. The efficiency of excitation energy capture by open photosystem II (PSII) (F(v)'/F(m)'), actual efficiency of PSII (Phi(PSII)), and the coefficient on photochemical quenching (qP) of plants with NaCl were reduced with time, and the reduction was alleviated till the control level by treatment with Put. The F(v)'/F(m)', Phi(PSII), and qP of plants without NaCl and/or with Put showed no variation during the experiment. Non-photochemical quenching of the singlet excited state of chlorophyll a (NPQ) showed quite different manner from the others as mentioned above, namely, continued to enhance during the experiment.