Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009. © 2009 Wiley‐Liss, Inc.     

SUN-1 and ZYG-12, Mediators of Centrosome–Nucleus Attachment,Are a Functional SUN/KASH Pair in Caenorhabditis elegans

Klarsicht/ANC-1/Syne/homology (KASH)/Sad-1/UNC-84 (SUN) protein pairs can act as connectors between cytoplasmic organelles and the nucleoskeleton. Caenorhabditis elegans ZYG-12 and SUN-1 are essential for centrosome–nucleus attachment. Although SUN-1 has a canonical SUN domain, ZYG-12 has a divergent KASH domain. Here, we establish that the ZYG-12 mini KASH domain is functional and, in combination with a portion of coiled-coil domain, is sufficient for nuclear envelope localization. ZYG-12 and SUN-1 are hypothesized to be outer and inner nuclear membrane proteins, respectively, and to interact, but neither their topologies nor their physical interaction has been directly investigated. We show that ZYG-12 is a type II outer nuclear membrane (ONM) protein and that SUN-1 is a type II inner nuclear membrane protein. The proteins interact in the luminal space of the nuclear envelope via the ZYG-12 mini KASH domain and a region of SUN-1 that does not include the SUN domain. SUN-1 is hypothesized to restrict ZYG-12 to the ONM, preventing diffusion through the endoplasmic reticulum. We establish that ZYG-12 is indeed immobile at the ONM by using fluorescence recovery after photobleaching and show that SUN-1 is sufficient to localize ZYG-12 in cells. This work supports current models of KASH/SUN pairs and highlights the diversity in sequence elements defining KASH domains.     

Purification and Functional Characterisation of Rhiminopeptidase A,a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

Background

Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros.

Methodology and Principal Findings

The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped β domain, a mixed α and β catalytic domain, a β-sandwich domain and a C-terminal α helical domain.

Conclusions

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.     

Epidemiologic Investigation of Immune-Mediated Polyradiculoneuropathy among Abattoir Workers Exposed to Porcine Brain

Background

In October 2007, a cluster of patients experiencing a novel polyradiculoneuropathy was identified at a pork abattoir (Plant A). Patients worked in the primary carcass processing area (warm room); the majority processed severed heads (head-table). An investigation was initiated to determine risk factors for illness.

Methods and Results

Symptoms of the reported patients were unlike previously described occupational associated illnesses. A case-control study was conducted at Plant A. A case was defined as evidence of symptoms of peripheral neuropathy and compatible electrodiagnostic testing in a pork abattoir worker. Two control groups were used - randomly selected non-ill warm-room workers (n = 49), and all non-ill head-table workers (n = 56). Consenting cases and controls were interviewed and blood and throat swabs were collected. The 26 largest U.S. pork abattoirs were surveyed to identify additional cases. Fifteen cases were identified at Plant A; illness onsets occurred during May 2004–November 2007. Median age was 32 years (range, 21–55 years). Cases were more likely than warm-room controls to have ever worked at the head-table (adjusted odds ratio [AOR], 6.6; 95% confidence interval [CI], 1.6–26.7), removed brains or removed muscle from the backs of heads (AOR, 10.3; 95% CI, 1.5–68.5), and worked within 0–10 feet of the brain removal operation (AOR, 9.9; 95% CI, 1.2–80.0). Associations remained when comparing head-table cases and head-table controls. Workers removed brains by using compressed air that liquefied brain and generated aerosolized droplets, exposing themselves and nearby workers. Eight additional cases were identified in the only two other abattoirs using this technique. The three abattoirs that used this technique have stopped brain removal, and no new cases have been reported after 24 months of follow up. Cases compared to controls had higher median interferon-gamma (IFNγ) levels (21.7 pg/ml; vs 14.8 pg/ml, P<0.001).

Discussion

This novel polyradiculoneuropathy was associated with removing porcine brains with compressed air. An autoimmune mechanism is supported by higher levels of IFNγ in cases than in controls consistent with other immune mediated illnesses occurring in association with neural tissue exposure. Abattoirs should not use compressed air to remove brains and should avoid procedures that aerosolize CNS tissue. This outbreak highlights the potential for respiratory or mucosal exposure to cause an immune-mediated illness in an occupational setting.     

Ultrastructural identification of non-adrenergic,non-cholinergic nerves in the rat anococcygeus muscle

The innervation of the rat anococcygeus muscle has been investigated ultrastructurally following fixation with a modified chromaffin reaction for the demonstration of biogenic amines (Tranzer and Richards, 1976). Three types of nerve profiles were revealed: (1) 60-70% of the profiles are adrenergic; (2) less than 5% of the profiles appear to be cholinergic; (3) up to 40% of the profiles are distinguished by the presence of a characteristically high proportion of electron-opaque, chromaffin-negative vesicles, 85-110 nm in diameter. This third type of profile was not affected by 6-OHDA, and is considered to represent the non-adrenergic, non-cholinergic inhibitory innervation of this tissue. Because of the morphological similarity of this nerve type, apart from the smaller vesicle size, to classical peptidergic nerve endings, they have been termed "small p-type" (sp-type). These results are discussed in relation to a previous report describing only two types of nerve profiles in this tissue (Gillespie and Lüllmann-Rauch, 1974).     

Alcohol and acetaldehyde metabolism in Caucasians,Chinese and Amerinds.

Ethanol (0.4 to 0.8 g/kg in 30 minutes) was given by mouth to 102 healthy young volunteers (37 Caucasian men, 21 Caucasian women, 20 Chinese men and 24 Ojibwa men). Venous blood concentrations of ethanol and acetaldehyde 60, 90, 120 and 150 minutes after the end of drinking were measured by gas chromatography. The calculated rates of ethanol metabolism in the Caucasian men and women did not differ, but the overall group means for subgroups of Caucasians (103.6 mg/kg-h), Chinese (136.6 mg/kg-h) and Ojibwa (182.7 mg/kg-h) with decreasing postabsorption values differed significantly from each other. Mean acetaldehyde values paralleled the rates of ethanol metabolism: Ojibwa, 14.6 mug/ml; Chinese, 10.0 mug/ml; and Caucasians, 9.4 mug/ml. The high rate of ethanol metabolism in Amerind subjects differs from previous findings. Habitual level of alcohol consumption, proportion of body fat and genetic factors appear to account for most of the group differences.     

Lack of correlation between ultrastructural and pharmacological types of non-adrenergic autonomic nerves

Summary In order to test the premise that non-adrenergic, non-cholinergic (NANC) autonomic nerves have a distinctive ultrastructural appearance, clearly different from that of cholinergic nerves, a detailed quantitative ultrastructural analysis has been made of the non-adrenergic innervation of 15 tissues thought from pharmacological evidence to be innervated by NANC nerves (rat and rabbit anococcygeus muscles; rabbit hepatic portal vein; extrinsically denervated toad lung); cholinergic nerves (atria of rat, rabbit, guinea-pig and toad); or both (guinea-pig cervical and thoracic trachealis muscle; rabbit rectococcygeus muscle; urinary bladder of rat, rabbit, guinea-pig and toad) in addition to their adrenergic supply. Following fixation with a modified chromaffin procedure allowing identification of adrenergic nerves, large, randomly selected samples of non-adrenergic nerve profiles from each tissue were analysed with respect to numbers, relative proportions, and size frequency distributions of different vesicle classes within the profiles. The neuromuscular relationships within each tissue were also analysed. On the basis of these analyses, it is clear that there are no consistent ultrastructural differences between cholinergic and NANC autonomic nerves: neither proportions nor sizes of the vesicles provide any clue as to the transmitter used by a particular nerve. The great majority of nerve profiles, whether cholinergic or NANC, contain predominantly small clear synaptic vesicles. Large filled peptidergic vesicles are no more common in most NANC nerves than in most cholinergic ones. It is concluded, on ultrastructural grounds, that the primary transmitter in these NANC autonomie nerves is most likely to be stored in and released from the small clear vesicles.     

Germline chimeric chickens from FACS‐sorted donor cells

Germline chimeric chickens can be constructed by injecting donor chicken blastodermal cells (CBCs) into recipient embryos and incubating to hatch. Transgenic chickens can be produced through chimeric intermediates if the donor cells are genetically manipulated; the chance of producing a transgenic chimera would be increased by enriching the donor population in transfected cells. To demonstrate that donor CBCs can be sorted according to the expression of a foreign gene, CBCs in suspension were subjected to transfection with plasmid DNA encoding bacterial β‐galactosidase (β‐gal). Following an overnight incubation, the CBCs were loaded with 5‐dodecanoylaminofluorescein di‐β‐D‐galactopyranoside (C₁₂FDG), which is fluorescent after cleavage by β‐gal. The treated cells were subjected to fluorescence activated cell sorting (FACS) to give “positive” (fluorescent) and “negative” (non‐fluorescent) populations. Almost 100% of the “positive” population showed β‐gal activity. “Positive” cells were cultured on mouse SNL 76/7 fibroblast feeder cells and formed colonies, most of which still stained positively for β‐gal activity after three days. FACS‐sorted cells of Barred Plymouth Rock origin were injected into recipient White Leghorn embryos, resulting in chimeric embryos. Of the 298 embryos injected with sorted cells, 23 (8%; 18 injected with “positive cells, five with “negative”) survived to rearing. Somatic chimerism was seen in 12 of 18 (67%) “positive” and three of five (60%) “negative” birds with the proportion of black pigmentation averaging 19% overall. Twenty birds reached sexual maturity, of which 12 (60%) were somatically chimeric; seven (35%) of these produced donor‐derived chicks. Donor CBCs can, therefore, be sorted by FACS according to the expression of a selectable marker gene without impairing their ability to contribute to germline chimeras; this procedure could be incorporated into a practicable method by which to increase the chances of producing a transgenic chicken. Mol. Reprod. Dev. 52:33–42, 1999. © 1999 Wiley‐Liss, Inc.     

Seasonal Rotifer Dynamics in the Long-term (1969–2002) Record from Lake Kinneret (Israel)

Long-term (1969–2002) data record of biomass distribution of rotifers in Lake Kinneret is combined with previously published information on their metabolic activity and newly calculated population dynamics parameters to synthesize a model of their seasonal dynamics in Lake Kinneret. Nineteen rotifer species were recorded in routine samples collected in Lake Kinneret (Israel) in 7 offshore (deeper than 5 m), stations, at 12 discrete depths during 1969–2002. Organisms were sorted and counted (including external egg carrying females), biomass was measured and calculated for the entire lake stock (g_w.w m⁻²; mg l⁻¹). Rates of grazing, respiration and production were measured experimentally at three different temperature ranges. Results were extrapolated to the lake community for months with similar temperatures. Rotifera comprised 7% of total zooplankton biomass in Lake Kinneret whilst Cladocera and Copepoda 58 and 35% respectively. Rotifers were found to be more abundant during December–June and decline in summer months. Monthly (1969–2001) means indicated total grazing capacity of rotifers as 11%, respiration as 9% and production as 3.7% of the total zooplankton metabolic activity. Positive relations were indicated between rotifer and small bodied cladoceran numerical concentrations. Population growth models suggest that rotifers are not food limited in Lake Kinneret but that fish predation plays an important role in regulating abundance in spring-summer and fall.