Cloning and sequencing of the gene encoding an aldehyde dehydrogenase that is induced by growing Alteromonas sp. Strain KE10 in a low concentration of organic nutrients

The protein composition of Alteromonas sp. strain KE10 cultured at two different organic-nutrient concentrations was determined by using two-dimensional polyacrylamide gel electrophoresis. The cellular levels of three proteins, OlgA, -B, and -C, were considerably higher in cells grown in a low concentration of organic nutrient medium (LON medium; 0.2 mg of carbon per liter) than cells grown in a high concentration of organic nutrient medium (HON; 200 mg of C liter(-1)) or cells starved for organic nutrients. In the LON medium, the cellular levels of the Olg proteins were higher at the exponential growth phase than at the stationary growth phase. A sequence of the gene for OlgA revealed that the amino acid sequence had a high degree of similarity to the NAD(+)-dependent aldehyde dehydrogenases of several bacteria. OlgA, expressed in Escherichia coli, catalyzed the dehydrogenation of acetaldehyde, propionaldehyde, and butyraldehyde. The aldehyde dehydrogenase activity of KE10 was higher in cells growing exponentially in LON medium than in HON. OlgA may be involved in the growth under low-nutrient conditions. The physiological role of OlgA is discussed here.     

Scrotal cutaneous verruciform xanthoma with monocyte chemoattractant protein-1 immunohistochemical study: a case report

ABSTRACT: INTRODUCTION: Verruciform xanthoma is a rare, benign lesion characterized by hyperkeratosis and aggregates of foam cell macrophages. Here, we describe a case of verruciform xanthoma on the scrotum, in which the immunohistochemical localization of monocyte chemoattractant protein-1, a chemokine of the C-C or beta family that has been shown to induce the recruitment of monocytes for injured tissue, was analyzed to determine which cells release chemoattractants for macrophages. CASE PRESENTATION: A 75-year-old Japanese man with a well-defined nodule on the left scrotum was admitted to the hospital. An excision biopsy revealed epidermal papillary proliferation with parakeratosis, hyperkeratosis, and infiltration of foam cell macrophages, whereby a pathological diagnosis of benign cutaneous verruciform xanthoma was made. Immunohistochemically, monocyte chemoattractant protein-1 was observed predominantly on cytokeratin AE1/AE3-positive differentiating keratinocytes in the prickle cell layer. However, while infiltrating macrophages were densely stained for monocyte chemoattractant protein-1, keratinocytes in the basal and parabasal layers were almost negative. CONCLUSIONS: We demonstrated that keratinocyte-derived monocyte chemoattractant protein-1 plays an important role in the establishment of particular histological features of verruciform xanthoma. However, in the present case, unlike in previous reports, monocyte chemoattractant protein-1 immunostaining in keratinocytes in the basal and parabasal layers was not prominent. We speculate that in the active phase of verruciform xanthoma, when continuous stimuli that release monocyte chemoattractant protein-1 from keratinocytes to the surrounding stromal area are present, the apparent immunostaining of monocyte chemoattractant protein-1 can be underestimated because of the void created by accelerated keratinocyte release from the cytoplasmic fraction.     

Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.     

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.     

Exercise pressor reflex function in female rats fluctuates with the estrous cycle

In women, sympathoexcitation during static handgrip exercise is reduced during the follicular phase of the ovarian cycle compared with the menstrual phase. Previous animal studies have demonstrated that estrogen modulates the exercise pressor reflex, a sympathoexcitatory mechanism originating in contracting skeletal muscle. The present study was conducted in female rats to determine whether skeletal muscle contraction-evoked reflex sympathoexcitation fluctuates with the estrous cycle. The estrous cycle was judged by vaginal smear. Plasma concentrations of estrogen were significantly (P < 0.05) higher in rats during the proestrus phase of the estrus cycle than those during the diestrus phase. In decerebrate rats, either electrically induced 30-s continuous static contraction of the hindlimb muscle or 30-s passive stretch of Achilles tendon (a maneuver that selectively stimulates mechanically sensitive muscle afferents) evoked less renal sympathoexcitatory and pressor responses in the proestrus animals than in the diestrus animals. Renal sympathoexcitatory response to 1-min intermittent (1- to 4-s stimulation to relaxation) bouts of static contraction was also significantly less in the proestrus rats than that in the diestrus rats. In ovariectomized female rats, 17β-estradiol applied into a well covering the dorsal surface of the lumbar spinal cord significantly reduced skeletal muscle contraction-evoked responses. These observations demonstrate that the exercise pressor reflex function and its mechanical component fluctuate with the estrous cycle in rats. Estrogen may cause these fluctuations through its attenuating effects on the spinal component of the reflex arc.     

Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum

The Manila clam Ruditapes philippinarum is infected with 2 Perkinsus species, Perkinsus olseni and P. honshuensis, in Japan. The latter was described as a new species in Mie Prefecture, Japan, in 2006. Ray's Fluid Thioglycollate Medium (RFTM) assay has been most commonly used to quantify Perkinsus infection. However, this assay cannot discriminate between species that resemble one another morphologically. We developed real-time PCR assays for the specific quantification of P. olseni and P. honshuensis. DNA was extracted using Chelex resin. Cultured P. olseni and P. honshuensis cells were counted and spiked into uninfected clam gill tissue prior to DNA extraction to generate standard curves, which allowed quantification based on the PCR cycle threshold values. We compared the RFTM assay with both real-time PCR assays by quantifying Perkinsus spp. in gill tissue samples from the same individual clams obtained from various localities in Japan. Infection intensities estimated by both assays were significantly correlated (r2 = 0.70). Our results suggest that the prevalence and infection intensity of P. honshuensis are much lower than for P. olseni in Manila clams.     

Beneficial effect of beraprost sodium plus telmisartan in the prevention of arterial stiffness development in elderly patients with hypertension and cerebral infarction

Beraprost sodium (BPS, an analogue of prostacyclin) and telmisartan (TS, an angiotensin receptor blocker) have been reported to have a preventive effect on arterial stiffness in patients with cardiovascular diseases. The purpose of this study was to estimate the effects of a combined therapy using BPS and TS on arterial pulse wave velocity (PWV) values in elderly patients with hypertension and cerebral infarction. Over a 3-month period, 80 subjects with hypertension and histories of cerebral infarction received BPS only (120 microg/day p.o.), TS only (40 mg/day p.o.), both BPS and TS, or no medication at all (control). Arterial PWV and ankle brachial indices (ABI) were determined prior to and after 3 months of drug administration. During the follow-up, there were no significant changes in any of the parameters monitored with the exception of a significant decrease in systolic blood pressure in the BPS only, TS only, and BPS plus TS groups when compared to controls. The difference values for PWV in the control group, BPS only group, TS only group, and BPS plus TS group were +232.5, -114.6, -151.5, and -248.1 cm/s, respectively. The reduction values were significantly more pronounced in the BPS plus TS group than in the BPS only (P=0.037) and the TS only (P=0.022) groups. When BPS is combined with TS, an overall additive effect is seen in the improvement of PWV in Japanese patients with hypertension and cerebral infarction. This combination therapy is more beneficial than the corresponding monotherapies.