The Del1 deposition domain can immobilize 3α-hydroxysteroid dehydrogenase in the extracellular matrix without interfering with enzymatic activity

Developing methods that result in targeting of therapeutic molecules in gene therapies to target tissues has importance, as targeting can increase efficacy and decrease off target-side-effects. Work from my laboratory previously showed that the extracellular matrix protein Del1 is organized in the extracellular matrix (ECM) via the Del1 deposition domain (DDD). In this work, a fusion protein with DDD was made to assay the ability to immobilize an enzyme without disrupting enzymatic function. A prostatic cancer-derived cell line LNCap that grows in an androgen-dependent manner was used with 3α-hydroxysteroid dehydrogenase (3 αHD), which catalyzes dihydrotestosterone (DHT). Plasmids encoding a 3αHD:DDD fusion were generated and transfected into cultured cells. The effects of 3αHD immobilized in the ECM by the DDD were evaluated by monitoring growth of LNCap cells and DHT concentrations. It was demonstrated that the DDD could immobilize an enzyme in the ECM without interfering with function.     

Sequence analysis of HLA class II domains: characterization of the DQw3 family of DQB genes

HLA class II allelic variants within the DQw3-related family of genes carry distinct allo-specificities and have been implicated in specific HLA-disease associations, such as insulin-dependent diabetes mellitus. To investigate the nucleotide variations which characterize DQw3 genes, we applied a novel cDNA cloning strategy that uses a single-stranded vector/primer system to facilitate DNA sequencing of allelically variable gene families. Using a DQB-specific primer sequence and M13 bacteriophage as the cloning vector, direct cloning and sequencing of multiple DQB genes was performed without the need for second strand synthesis or for subcloning. Sequence analysis from eight lymphoblastoid cell lines selected to represent different ethnic backgrounds revealed three DQw3-related DQB genes, DQB3.1, 3.2, and 3.3, corresponding to the newly designated HLA-DQw7, w8, and w9 specificities, respectively. An unusual Pro-Pro couplet at codons 55–56 is characteristic of all DQw3-positive sequences and may be contributing to the broad DQw3 allospecificity. Comparisons among ethnically disparate DQw3-related sequences showed no additional expressed or silent nucleotide substitutions among these DQB alleles. Thus, polymorphism within the DQw3 family of genes appears to be extremely limited, with a paucity of nucleotide variations accumulated by evolutionary distance.     

Loss of symbiont infectivity following thermal stress can be a factor limiting recovery from bleaching in cnidarians

Increases in seawater temperature can cause coral bleaching through loss of symbiotic algae (dinoflagellates of the family Symbiodiniaceae). Corals can recover from bleaching by recruiting algae into host cells from the residual symbiont population or from the external environment. However, the high coral mortality that often follows mass-bleaching events suggests that recovery is often limited in the wild. Here, we examine the effect of pre-exposure to heat stress on the capacity of symbiotic algae to infect cnidarian hosts using the Aiptasia (sea-anemone)-Symbiodiniaceae model system. We found that the symbiont strain Breviolum sp. CS-164 (ITS2 type B1), both free-living and in symbiosis, loses the capacity to infect the host following exposure to heat stress. This loss of infectivity is reversible, however, a longer exposure to heat stress increases the time taken for reversal. Under the same experimental conditions, the loss of infectivity was not observed in another strain Breviolum psygmophilum CCMP2459 (ITS2 type B2). Our results suggest that recovery from bleaching can be limited by the loss of symbiont infectivity following exposure to heat stress.Subject terms: Microbial ecology, Biodiversity

Cnidarians including reef-building corals harbor endosymbiotic dinoflagellates of the family Symbiodiniaceae, from which they derive the majority of their energy. Therefore, the breakdown of the symbiotic relationship, a process known as bleaching, can result in the host starving. However, bleaching is not always lethal because symbiont densities can recover [1, 2]. Recovery from bleaching is driven mainly by symbiotic algae that remain within the bleached corals (the residual population) dividing and spreading throughout the colony [3], and also possibly through the recruitment of free-living symbiotic algae from the external environment [4]. In the last few decades, coral cover has drastically decreased in many regions, due to frequent mass coral bleaching events caused by global warming [5], implying that recovery from bleaching is often limited by unknown factors. In the present study, we demonstrate that both free-living and residual symbiont cells lose their capacity to infect cnidarian host cells once they are exposed to high temperature stress, and present this mechanism as a limiting factor for the host’s recovery from bleaching.We first examined the effect of pre-exposure to high temperature on infectivity using aposymbiotic Exaiptasia pallida (or “Aiptasia”) polyps (Supplementary Fig. 1) and cultured strains of Breviolum sp. CS-164 (ITS2 type B1). Symbiotic algae and polyps were separately incubated at either 25 or 32 °C for 3 days. Following this initial treatment, polyps were inoculated with symbiotic algae at 25 °C for 3 days. Infectivity was then determined by counting the number of algae in the tentacles where algal colonization occurs quickly and individual symbiont cells are easily visualized [6]. When both symbiotic algae and hosts were pre-exposed to 25 °C, significant numbers of algae were seen in tentacles (Fig. 1a). In contrast, the number (Fig. 1a) and density (Fig. 1b) of algae in the tentacles were significantly lower when both the algae and the host were exposed simultaneously to 32 °C, and when the algae alone were exposed to 32 °C. Thus, symbiotic algae, but not host polyps, lose their capacity to form a symbiotic relationship once they are exposed to high temperature. Neither cell viability measured by Evans blue staining (Supplementary Figs. 2a, b and 3) nor cell density (Supplementary Fig. 2c) differed between CS-164 cells exposed to 25 and 32 °C, indicating that infectivity was not lost by the lethal damage to cells. We repeated this experiment with another strain, B. psygmophilum CCMP2459 (ITS2 type B2). In contrast to the results with CS-164, high temperature had no effect on infectivity of CCMP2459 (Supplementary Fig. 4a). These results demonstrate that symbiotic algae can lose their capacity to infect host cells following exposure to high temperature and that thermal sensitivity differs between these two algal strains (Fig. 1b and Supplementary Fig. 4a).Open in a separate windowFig. 1Loss of infectivity in Breviolum sp. CS-164 following exposure to elevated temperature.a Fluorescent photographs of Aiptasia polyps 3 days after culturing with symbiont cells in four different treatments (i) neither symbionts nor polyps exposed to high temperature (32 °C) for 3 days, (ii) only symbionts exposed to high temperature, (iii) only polyps exposed to high temperature, (iv) both symbionts and polyps exposed to high temperature. Red dots show chlorophyll fluorescence from algal symbionts. b The density of symbionts in tentacles was measured 3 days after culturing Aiptasia polyps (H) with symbiont cells (S) in four different treatments, as indicated below the panel and outlined in the text. c The density of symbionts was measured 3 days after culturing Aiptasia polyps with symbiotic algae in different treatments. In this experiment, symbiotic algae that had been expelled from Aiptasia polyps cultured at 25 or 32 °C for 3 days were used to infect Aiptasia. b, c Values are log₂ fold changes with respect to the samples without any temperature treatment. Each point represents an independent experiment. ns, not significant (with p > 0.05); **, p < 0.01.We next examined whether the loss of infectivity following pre-exposure to high temperature also occurs when symbiotic algae are in symbiosis with the host rather than free living (Fig. 1c and Supplementary Fig. 4b). We prepared symbiotic Aiptasia polyps with either CCMP2459 or CS-164 by separately inoculating them in aposymbiotic polyps, and then exposed each group to either 25 or 32 °C for 3 days. Algae expelled from the polyps during this treatment were collected and then used to inoculate aposymbiotic Aiptasia at 25 °C. In CS-164, after 3 days of inoculation, symbiont density in Aiptasia became lower with algae collected at 32 °C than 25 °C (Fig. 1c). However, in CCMP2459, there was no difference in the infectivity between algae collected at 25 and 32 °C (Supplementary Fig. 4b). Our results demonstrate that symbiont cells, both free-living and in symbiosis, can lose infectivity following exposure to high temperature and that thermal sensitivity differs between these two algal strains.We then tested whether or not the temperature-induced loss of infectivity was reversible (Fig. 2). Free-living CS-164 cells were pre-exposed to 25 or 32 °C for either 2 or 3 days after which they were allowed to recover for a maximum of 10 days at 25 °C. After these treatments, symbiotic algae were used to inoculate aposymbiotic Aiptasia polyps at 25 °C for 3 days. Cells with pre-exposure to 32 °C for 2 days had lower infectivity but regained the capacity to infect host cells after a 5-day recovery period (Fig. 2). However, after 3 days exposure, infectivity gradually recovered but remained lower than the controls even after 10 days (Fig. 2). Our results demonstrate that the loss of infectivity following temperature stress is reversible in algal cells but a longer exposure to heat stress increases the time taken to reverse the loss of infectivity.Open in a separate windowFig. 2Reversibility of the lost infectivity upon the exposure to elevated temperature in Breviolum sp. CS-164.The density of symbionts in tentacles was measured following treatments as described in the text and shown by the relative to control. Values are log₂ fold changes with respect to the control. The box and line represent the quartiles and median, respectively. Each point represents an independent experiment. *, p < 0.05.In the present study, infectivity was tested by introducing symbiotic algae directly into the host’s body cavity, suggesting that the loss of infectivity seen in our experiments is likely due to a failure of the host to take up the algal cells via phagocytosis or for symbiont cells to persist within host cells. Factors such as symbiont cell size [7] and the symbiont surface glycome [8, 9] are potentially important determinants of symbiont uptake and persistence, though we still know little about this topic (see review [10]). Furthermore, it is unknown how these various discriminatory factors are influenced by thermal stress.In coral larvae and juveniles, the initiation of symbiosis with algae is reduced at high temperatures, suggesting that global warming will complicate the relationship between host and symbiont [11–13]. However, the mechanism for this reduction in the rate of symbiosis establishment is not clear. Our results suggest that the loss of symbiont infectivity is one possible cause of this phenomenon.Recovery of symbiont densities following coral bleaching relies on a supply of symbiont cells either from within the host or from the external environment. Our results show that symbiotic algae, both free-living and symbiotic, lose the capacity to infect the host following exposure to high temperature stress (Fig. 1). This loss of infectivity is reversible but dependent on the duration of the thermal stress (Fig. 2). Thus, following coral bleaching events, especially those induced by thermal anomalies that can last for weeks, symbiont densities within the host are unlikely to recover in time to avoid the host starving due to physiological compromise of the symbionts, rather than the host. Nonetheless, given the differences in sensitivity between two strains tested (Fig. 1b and Supplementary Fig. 4a), if heat tolerant symbionts are available in the environment, this might provide a chance for recovery.     

Simple enzymatic detection method for urinary sulfated 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7α-hydroxy bile acids, are detected by an enzymatic method using 7α-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7α-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by β-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7α-hydroxy bile acids are detected with 7α-HSD in the presence of β-NAD⁺, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7α-hydroxy bile acids may be useful for clinical diagnosis.     

Alpha-2-HS-glycoprotein phenotype frequencies in Cook Islanders

The polymorphism of the alpha 2-HS-glycoprotein (A2HS) was analysed in Rarotonga and Mangaia, the Cook Islands. The A2HS*2 frequency was found to be the highest value among all populations studied up to now. There was a significant difference in A2HS*2 gene frequencies between the two populations, Rarotonga (0.62) and Mangaia (0.76).     

A newly-discovered population ofColobus angolensis in East Africa

A very isolated population ofColobus angolensis has been newly discovered in Tanzania. The monkey has white, long epaulettes and cheek hair, but no white pygal band. Its tail has somewhat of a brushy tip, and is grey in color. This may very well be a heretofore undescribed, and distinct subspecies.     

The number of chlorophyll-less spots and the primary leaf area as criteria of radiation damage in soybean (Glycine max merill)

The number of chlorophyll-less spots occurring on the primary leaves as well as the primary leaf size were investigated in two soybean cultivars, differing genetically in radiosensitivity, after irradiation of seed with ⁶⁰Co γ-rays. A high correlation was found between increasing number of spots, decreasing leaf size and seedling growth inhibition.The number of spots can be used to monitor radiation effects over the small dose range where the growth inhibition is not pronounced. Primary leaf size can be used as a convenient criterion of seedling growth inhibition. Possible causes of leaf spotting are discussed.     

Expression of high activity of ornithine decarboxylase and occurrence of unusual chromophobic cells in anterior pituitary gland of a novel growth-retarded strain of mice, grm/grm

Extremely high activity of ornithine decarboxylase (ODC) was detected in the pituitary gland of growth- retarded mice, grm/grm at 2 months after birth. The elevated enzyme activity gradually decreased to the control level in 14 months after birth. In the pituitary gland of the growth-retarded mice, unusual chromophobic cells were also present from the early stages after birth. The chromophobic cells showed conspicuous proliferations and resulted in a distinct hyperplasia of the tissue after 4 months after birth. These findings suggest that OCD is correlated to the progressive transformation of pituitary cells into the chromophobic cells.