Development of calcareous skeletal elements in invertebrates

Most metazoans require skeletal support systems. While the formation of bones and teeth in vertebrates has been well studied, endo- and exoskeleton development of non-vertebrates, especially calcification during terminal differentiation, has been neglected. Biomineralization of skeletons in invertebrates presents interesting research opportunities. We undertake here to survey some of the better understood examples of skeletal development in selected invertebrates. The differentiation of the skeletal spicules of euechinoid larvae and other non-vertebrate deuterostomes, the shells of molluscs, and the calcification of crustacean carapaces are surveyed. The diversity of these different kinds of animals and our present limited understanding make it difficult to identify unifying themes, but there certainly are unifying questions: How is the mineral precursor secreted? What is the nature of the interaction of mineral with the matrix proteins of the skeleton? Is there any conservation of protein domains in matrix proteins found in skeletal elements from different phyla? Are there common strategies in the development of organs that form mineralized structures?     

A Novel Antibody against Human Properdin Inhibits the Alternative Complement System and Specifically Detects Properdin from Blood Samples

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1–10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H₂O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80–125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13–30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.     

Housekeeping genes in cancer: normalization of array data

Biological maintenance of cells under variable conditions should affect gene expression of only certain genes while leaving the rest unchanged. The latter, termed "housekeeping genes," by definition must reflect no change in their expression levels during cell development, treatment, or disease state anomalies. However, deviations from this rule have been observed. Using DNA microarray technology, we report here variations in expression levels of certain housekeeping genes in prostate cancer and a colorectal cancer gene therapy model system. To highlight, differential expression was observed for ribosomal protein genes in the prostate cancer cells and beta-actin in treated colorectal cells. High-throughput differential gene expression analysis via microarray technology and quantitative PCR has become a common platform for classifying variations in similar types of cancers, response to chemotherapy, identifying disease markers, etc. Therefore, normalization of the system based on housekeeping genes, such as those reported here in cancer, must be approached with caution.     

Removing the threat of diclofenac to critically endangered Asian vultures

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genusGyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captiveGyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vultureGyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered AsianGyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40G. africanus. Subsequently, sixG. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species(Gyps bengalensis,Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity toGyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India.     

Importance of hydration for gramicidin-induced hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes

The macroscopic organization, lipid head group conformation, and structural and dynamic properties of 2H2O were investigated in dioleoylphosphatidylcholine (DOPC) model systems of varying gramicidin and 2H2O (or H2O) content by means of small-angle X-ray diffraction and 31P and 2H NMR. At low stages of hydration, N less than 6 (N = 2H2O/DOPC molar ratio), a single lamellar phase is observed in which the gramicidin molecules become preferentially hydrated upon increasing N. For 6 less than N less than 12 phase separation occurs between a gramicidin-poor and a gramicidin-rich lamellar phase. This latter phase is characterized by a smaller repeat distance and decreased DOPC head group order. For N greater than 12, the gramicidin-rich lamellar phase converts to a hexagonal HII phase. Thus, hydration of gramicidin is a prerequisite for HII phase formation in the DOPC/gramicidin system. The HII phase is very rich in gramicidin and 2H2O (gramicidin:DOPC:H2O = 1:1.1:0.9 w/w/w). A model is proposed in which self-assembly of hydrated gramicidin molecules into domains of a specific structure plays a determinant role in the formation of the HII phase by gramicidin.     

Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation.

125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.     

Inspiratory muscle forces and endurance in maximum resistive loading

The ability of the respiratory muscles to sustain ventilation against increasing inspiratory resistive loads was measured in 10 normal subjects. All subjects reached a maximum rating of perceived respiratory effort and at maximum resistance showed signs of respiratory failure (CO2 retention, O2 desaturation, and rib cage and abdominal paradox). The maximum resistance achieved varied widely (range 73-660 cmH2O X l-1 X s). The increase in O2 uptake (delta Vo2) associated with loading was linearly related to the integrated mouth pressure (IMP): delta Vo2 = 0.028 X IMP + 19 ml/min (r = 0.88, P less than 0.001). Maximum delta Vo2 was 142 ml/min +/- SD 68 ml/min. There were significant (P less than 0.05) relationships between the maximum voluntary inspiratory pressure against an occluded airway (MIP) and both maximum IMP (r = 0.80) and maximum delta Vo2 (r = 0.76). In five subjects, three imposed breathing patterns were used to examine the effect of different patterns of respiratory muscle force deployment. Increasing inspiratory duration (TI) from 1.5 to 3.0 and 6.0 s, at the same frequency of breathing (5.5 breaths/min) reduced peak inspiratory pressure and increased the maximum resistance tolerated (190, 269, and 366 cmH2O X l-1 X s, respectively) and maximum IMP (2043, 2473, and 2913 cmH2O X s X min-1, but the effect on maximum delta Vo2 was less consistent (166, 237, and 180 ml/min). The ventilatory endurance capacity and the maximum O2 uptake of the respiratory muscles are related to the strength of the inspiratory muscles, but are also modified through the pattern of force deployment.