Cloning of the cDNA and gene for mouse mast cell protease 4. Demonstration of its late transcription in mast cell subclasses and analysis of its homology to subclass-specific neutral proteases of the mouse and rat

Based on the amino-terminal amino acid sequence of the mature form of mouse mast cell protease 4 (MMCP-4), previously identified in peritoneal connective tissue mast cells (CTMC) and Kirsten sarcoma virus-immortalized mast cells (KiSV-MC), a 26-mer oligonucleotide probe was constructed and used to clone cDNAs for MMCP-4 from a KiSV-MC1 cDNA library. MMCP-4 is the first secretory granule serine protease of CTMC to be molecularly cloned. Using a cDNA probe derived from the 3'-untranslated portion of the MMCP-4 cDNA, the gene for MMCP-4 and a second highly related gene (mouse mast cell protease-like, MMCP-L) were cloned from a BALB/c mouse genomic DNA library and sequenced entirely, including approximately 2 kilobases of the 5'-flanking region. MMCP-4 and MMCP-L have five exons of identical length, four introns of nearly identical length, and approximately 900 base pairs of 5'-flanking DNA with sequence similarity by dot matrix analysis. By RNA blot analysis with gene-specific probes for MMCP-4 (bases 497-633 of the cDNA) and MMCP-L (bases 502-638 of the cDNA), mRNA for MMCP-4 was present in KiSV-MC5, CTMC, and the intestine of a mouse infected with the parasite Nippostrongylus brasiliensis markedly enriched for mucosal mast cells (MMC); MMCP-L mRNA was detected only in the intestine of the N. brasiliensis-infected mouse. MMCP-4 mRNA was not expressed in normal mouse intestine or in interleukin 3-dependent bone marrow-derived mast cells, which can serve as precursors to both MMC and CTMC. This finding suggests that MMCP-4 is transcribed relatively late in the development of both the CTMC and MMC subclasses and underscores the fact that mouse bone-marrow-derived mast cells are immature mast cells, rather than tissue culture equivalents of the MMC subclass.     

Xanthomicrol is the main cytotoxic component of Dracocephalum kotschyii and a potential anti-cancer agent

Spinal-Z, a methanolic mixture of dried powdered seeds of Peganum harmala Linn. and leaf of Dracocephalum kotschyii Boiss. is an Iranian ethno-medical remedy. It has been used for the treatment of various types of cancer for many years. To evaluate the use of Spinal-Z in treatment of cancer, we examined its effects against a panel of malignant cell lines and tumors induced in mice. The in vitro antiproliferative activities of Spinal-Z, the seed extract of P. harmala and the leaf extract of D. kotschyii were determined using the MTT assay. The concentration of the agent required to inhibit cell growth by 50% (IC50) was estimated. In addition, the anti-tumor activities of the remedy and its constituents were investigated. Viability of cells treated with Spinal-Z and its components decreased in a dose dependent manner. Spinal-Z and its components showed cytotoxic effects against all cell lines tested. The leaf extract of D. kotschyii showed a greater preferential cytotoxic effect than the seed extract of P. harmala and Spinal-Z, on all cell lines tested. Harmine showed cytotoxicity against HL60 and K562 cell lines. This could explain the cytotoxic effect of P. harmala on these cells. The leaf extract of D. kotschyii was able to inhibit tumor proliferation in mice. The active ingredient in the leaf extract of D. kotschyii appears to be a flavone identified as xanthomicrol. Xanthomicrol was able to inhibit proliferation of a number of malignant cells. The cytotoxic effects of xanthomicrol were more selective towards malignant cells than doxorubicin.     

Serum copper and zinc concentrations are lower in Iranian patients with angiographically defined coronary artery disease than in subjects with a normal angiogram.

BACKGROUND: An imbalance between zinc and copper metabolism has been reported to predispose to coronary artery disease (CAD) in Western populations, but there are little data for other racial groups. We have therefore investigated the association between serum copper and zinc, and CAD in Iranian subjects undergoing coronary angiography. METHODS: Serum copper, zinc, fasting lipid profile, and blood glucose levels were measured in 114 patients (67 male and 47 female) undergoing routine coronary angiogram. Anthropometric features including blood pressure were determined using standard procedures. Demographic characteristics, including menopausal status and smoking habit, were assessed by questionnaire. RESULTS: Male patients had lower serum copper (p<0.05), lower serum zinc (p<0.05), and higher serum zinc/copper ratio (p<0.05) than females. Serum copper and zinc concentrations were significantly lower in the subjects with angiographically defined CAD than those patients with a normal angiogram, although the zinc/copper ratio was higher in these patients (p<0.001). Serum copper (r=-0.303, p<0.001) and zinc (r=-0.250, p<0.01) concentrations were both inversely related to age, and copper was positively associated with fasting serum triglycerides (r=0.188, p<0.05). CONCLUSION: Serum copper and zinc concentrations were significantly lower in Iranian patients with abnormal versus those with a normal angiogram. However, the zinc/copper ratio was higher in patients with CAD compared to subjects without CAD. Serum zinc and copper concentrations appear to be influenced by several physiological factors including age and gender.     

Analyzing Thermal Stability of Cell Membrane of Salmonella Using Time-Multiplexed Impedance Sensing

Heat treatment is one of the most widely used methods for inactivation of bacteria in food products. Heat-induced loss of bacterial viability has been variously attributed to protein denaturation, oxidative stress, or membrane leakage; indeed, it is likely to involve a combination of these processes. We examine the effect of mild heat stress (50–55°C for ≤12 min) on cell permeability by directly measuring the electrical conductance of samples of Salmonella enterica serovar Typhimurium to answer a fundamental biophysical question, namely, how bacteria die under mild heat stress. Our results show that when exposed to heat shock, the cell membrane is damaged and cells die mainly due to the leakage of small cytoplasmic species to the surrounding media without lysis (confirmed by fluorescent imaging). We measured the conductance change, ΔY, of wild-type versus genetically modified heat-resistant (HR) cells in response to pulse and ramp heating profiles with different thermal time constants. In addition, we developed a phenomenological model to correlate the membrane damage, cytoplasmic leakage, and cell viability. This model traces the differential viability and ΔY of wild-type and HR cells to the difference in the effective activation energies needed to permeabilize the cells, implying that HR cells are characterized by stronger lateral interactions between molecules, such as lipids, in their cell envelope.     

The Effects of Magnesium and Zinc Co-Supplementation on Biomarkers of Inflammation and Oxidative Stress,and Gene Expression Related to Inflammation in Polycystic Ovary Syndrome: a Randomized Controlled Clinical Trial

Magnesium and zinc are known to exert multiple beneficial effects including anti-inflammatory and antioxidant actions. To our knowledge, data on the effects of magnesium and zinc co-supplementation on biomarkers of inflammation and oxidative stress and gene expression related to inflammation in subjects of polycystic ovary syndrome (PCOS) are scarce. This study was conducted to evaluate the effects of magnesium and zinc co-supplementation on biomarkers of inflammation and oxidative stress and gene expression related to inflammation in subjects with PCOS. This randomized double-blind, placebo-controlled trial was conducted among 60 subjects with PCOS diagnosed according to the Rotterdam criteria, aged 18–40 years old. Participants were randomly assigned into two groups to take either 250 mg of magnesium oxide plus 220 mg of zinc sulfate (containing 50 mg zinc) supplements (n?=?30) or placebo (n?=?30) twice a day for 12 weeks. Biomarkers of inflammation and oxidative stress were assessed at baseline and at end of treatment. Gene expression related to inflammatory cytokines was assessed in peripheral blood mononuclear cells (PBMCs) of PCOS women with RT-PCR method. After the 12-week intervention, compared with the placebo, magnesium and zinc co-supplementation significantly decreased serum high-sensitivity C-reactive protein (hs-CRP) (??1.6?±?2.4 vs. +?0.1?±?0.7 mg/L, P?=?0.001) and protein carbonyl (PCO) (??0.14?±?0.28 vs. +?0.02?±?0.07 mmol/mg protein, P?=?0.002) and significantly increased plasma total antioxidant capacity (TAC) levels (+?60.7?±?69.4 vs. ??1.5?±?141.5 mmol/L, P?=?0.03). Results of RT-PCR demonstrated that compared with the placebo, magnesium and zinc co-supplementation downregulated gene expression of interleukin-1 (IL-1) (P?=?0.007) and tumor necrosis factor alpha (TNF-α) (P?=?0.03) in PBMCs of subjects with PCOS. Overall, magnesium and zinc co-supplementation, compared with the placebo, for 12 weeks among PCOS women had beneficial effects on serum hs-CRP, plasma PCO, TAC, and gene expression of IL-1 and TNF-α. Clinical trial registration number: http://www.irct.ir: IRCT201706075623N121.     

Gonad maturation and spawning of cobia,Rachycentron canadum (Linnaeus, 1766) off the Dungun coast,Malaysia

The reproductive biology of cobia, Rachycentron canadum, from the coastal waters of Dungun, Malaysia was studied from June 2014 to May 2015. From commercial trawls, a total of 201 samples (combined sexes) were collected (fork lengths [FL] 37.5–124.0 cm; body weights 0.5–20.4 g). The overall sex ratio of females to males was 1:0.9, which was not significantly different (χ² = 2.12, df = 1; p < .05). Estimations of length at 50% maturity (L₅₀) showed that both sexes matured at approximately 75 cm FL; estimated spawning frequency was 6 days. Mean batch fecundity (BF) ranged from 0.55 to 4.32 million eggs. The average number of eggs per gram of ovary was from 2,100 to 5,400 eggs, and relative fecundity 147 eggs/g. There was a weak positive correlation (r² = .48) between BF and female FL as well as BF with an ovary‐free body weight (r² = .56), possibly due to females being in a continuous spawning condition and some possibly half‐spent, based on the histological examination of the female gonads. Despite cobia being asynchronous spawners, the gonadosomatic index in both males and females showed peaks in June, November, and particularly March. Based on histological examination, spawning‐capable males were encountered throughout the study period, whereas spawning‐capable females in the late developing subphase were found mostly in March and April. This is the first study on the reproductive aspects of cobia in Malaysian waters.     

Towards the conservation of a critically endangered species, <Emphasis Type="Italic">Aphanius farsicus</Emphasis>: embryogenesis and development

Human-induced disturbance or anthropogenic activities, natural disturbance and also limited distribution, have forced Aphanius farsicus populations to the edge of extinction, during the past few years. As knowledge of fish ontogeny is fundamental for accurate stock assessment, and reproductive potential determination, thus for future conservation and management of the critically endangered species, we describe the first detailed normal embryonic development of this cyprinodontid fish from fertilization to post-hatching. Aphanius farsicus spontaneously spawned at 24°C. The newly-laid eggs were hyaline and spherical in shape (1.40 ± 0.02 mm, n = 11), sticky and full of egg yolk (telolecithal/macrolecithal) with oil droplets. We documented the main developmental stages at 24°C to zygote (0 h), cleavage (2 h), blastula (2 h), gastrula (17 h), segmentation, (34 h), pharyngula (100 h) and hatching (162 h).