Identification of Immunoreactive Material in Mammoth Fossils

The fossil record represents a history of life on this planet. Attempts to obtain molecular information from this record by analysis of nucleic acids found within fossils of extreme age have been unsuccessful or called into question. However, previous studies have demonstrated the long-term persistence of peptides within fossils and have used antibodies to extant proteins to demonstrate antigenic material. In this study we address two questions: Do immunogenic/antigenic materials persist in fossils? and; Can fossil material be used to raise antibodies that will cross-react with extant proteins? We have used material extracted from a well-preserved 100000-300000-year-old mammoth skull to produce antisera. The specificity of the antisera was tested by ELISA, western blotting, and immunohistochemistry. It was demonstrated that antisera reacted specifically with the fossils and not the surrounding sediments. Reactivity of antisera with modern proteins and tissues was also demonstrated, as was the ability to detect evolutionary relationships via antibody-antigen interactions. Mass spectrometry demonstrated the presence of amino acids and specific peptides within the fossil. Peptides were purified by anion-exchange chromatography and sequenced by tandem mass spectrometry. The collagen-derived peptides may have been the source of at least some of the immunologic reactivity, but the antisera identified molecules that were not observed by mass spectrometry, indicating that immunologic methods may have greater sensitivity. Although the presence of peptides and amino acids was demonstrated, the exact nature of the antigenic material was not fully clarified. This report demonstrates that antibodies may be used to obtain information from the fossil record.     

Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76+/-1.21 (arbitrary units) for males as compared to 3.37+/-1.47 for females (P<0.05)], tail DNA (%) [10.2+/-2.96 for males as compared to 9.40+/-2.83 for females (P<0.05)] and tail length (microm) [59.65+/-9.23 for males and 49.57+/-14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.     

How pH regulates a pH regulator: a regulatory hot spot in the N-terminal cytoplasmic domain of the AE2 anion exchanger

Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na⁺-independent chloride-bicarbonate exchanger genes and multiple Na⁺-bicarbonate cotransporter and Na⁺-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na⁺-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.     

Curcumin differentially regulates TGF-beta1, its receptors and nitric oxide synthase during impaired wound healing

Wound healing is a highly ordered process, requiring complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important. Nitric oxide is also an important factor in healing and its production is regulated by inducible nitric oxide synthase (iNOS). We have earlier shown that curcumin (diferuloylmethane), a natural product obtained from the plant Curcuma longa, enhances cutaneous wound healing in normal and diabetic rats. In this study, we have investigated the effect of curcumin treatment by topical application in dexamethasone-impaired cutaneous healing in a full thickness punch wound model in rats. We assessed healing in terms of histology, morphometry, and collagenization on the fourth and seventh days post-wounding and analyzed the regulation of TGF-beta1, its receptors type I (tIrc) and type II (tIIrc) and iNOS. Curcumin significantly accelerated healing of wounds with or without dexamethasone treatment as revealed by a reduction in the wound width and gap length compared to controls. Curcumin treatment resulted in the enhanced expression of TGF-beta1 and TGF-beta tIIrc in both normal and impaired healing wounds as revealed by immunohistochemistry. Macrophages in the wound bed showed an enhanced expression of TGF-beta1 mRNA in curcumin treated wounds as evidenced by in situ hybridization. However, enhanced expression of TGF-beta tIrc by curcumin treatment observed only in dexamethasone-impaired wounds at the 7th day post-wounding. iNOS levels were increased following curcumin treatment in unimpaired wounds, but not so in the dexamethasone-impaired wounds. The study indicates an enhancement in dexamethasone impaired wound repair by topical curcumin and its differential regulatory effect on TGF-beta1, it's receptors and iNOS in this cutaneous wound-healing model.     

Correction of defective early tyrosinase processing by bafilomycin A1 and monensin in pink-eyed dilution melanocytes

Mutations in the human P gene result in oculocutaneous albinism type 2, the most common form of albinism. Mouse melan-p1 melanocytes, cultured from mice null at the homologous pink-eyed dilution (p) locus, exhibit defective melanin production. A variety of compounds including tyrosine, NH4Cl, bafilomycin A1, concanamycin, monensin, and nigericin are capable of restoring melanin synthesis in these cells. In the current study, we investigated the subcellular effects of bafilomycin A1 and monensin treatment of melan-p1 cells. Both agents play two roles in the processing of tyrosinase (Tyr) in melan-p1 cells. First, combined glycosidase digestion and immunoblotting analysis showed that these agents reduce levels of Tyr retained in the endoplasmic reticulum (ER) and facilitate the release of Tyr from the ER to the Golgi. Secondly, treatment with these compounds resulted in the stabilization of Tyr. Surprisingly, induction of melanin synthesis corresponds more closely with diminution of ER-retained Tyr, rather than the absolute amount of Tyr. Our results suggest that bafilomycin A1 and monensin induce melanin synthesis in melan-p1 cells mainly by facilitating Tyr processing from the ER to the Golgi by increasing the pH in either the ER or the ER-Golgi intermediate compartment.     

The distribution and evolutionary history of Wolbachia infection in native and introduced populations of the invasive argentine ant (Linepithema humile)

Wolbachia pipientis is a maternally transmitted bacterium that often alters the life history of its insect host to maximize transmission to subsequent generations. Here we report on the frequency and distribution of Wolbachia infection in a widespread invasive species, the Argentine ant (Linepithema humile). We screened 1175 individual Argentine ants from 89 nests on five continents and several islands, including numerous locations in both the native (South American) and introduced ranges. We detected Wolbachia in four of 11 native populations, but only one of 21 introduced populations was infected. In the Argentine ant's native range, the distribution of Wolbachia supergroups A and B was nonoverlapping. By coupling infection frequency data with behaviourally defined colony boundaries, we show that infected and uninfected colonies are often adjacent to one another, supporting the proposition that little female-mediated gene flow occurs among Argentine ant colonies. We also conduct a phylogenetic analysis, and show that the Wolbachia infecting both native and introduced populations of Argentine ants belong to two lineages that appear to be specialized on infecting New World ants. One other lineage of Wolbachia has undergone frequent, recent episodes of horizontal transmission between distantly related, introduced insect hosts.     

The water load test: observations from healthy controls and patients with functional dyspepsia

Gastric sensation and accommodation are studied by barostat, but this is invasive. The drink test is noninvasive and may provide similar information. We evaluated relationships between drink test, gastric function, symptoms, and psychiatric distress. Controls (73) and functional dyspeptics (FD) (92) were studied using a 5-min water load test (WL5), gastric emptying, and electrogastrography (EGG). Symptoms, quality of life, and psychiatric distress were measured using standardized measures. Controls underwent test-retest of WL5 and comparison of WL5 with 100 ml/min water-based drink test (WL100) or nutrient drink. Controls, FD, and gastroparetics estimated drinking capacity before WL5 using a visual analog scale. WL5 correlated with WL100 (r = 0.7929) but not nutrient drink test (r = 0.1995). WL5 was significantly less in FD than controls, and abnormal WL5 was seen in 46%. In FD, volume to fullness inversely correlated with symptom severity (r =-0.29; P = 0.0154) and WL5 produced more symptoms, particularly nausea. Gastric function was not different between FD with normal or abnormal WL5. Symptoms and psychiatric distress were similar between normal and abnormal WL5 groups, but the abnormal group had significantly poorer quality of life. Controls and gastroparetics had good correlation of estimated and ingested volumes, but FD did not. Versus FD with normal WL5 capacity, FD with impaired drinking capacity have normal gastric function and similar symptoms but poorer quality of life. FD are less able to predict drinking capacity. These data suggest that WL5 identifies FD with intact gastric function but abnormal visceral perception.     

PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation

It has been known for over a decade that inhibition of protein phosphatase 1 (PP1) activity prevents entry into M phase, but the relevant substrate has not been identified. We report here that PP1 is required for dephosphorylation of the Cdc2-directed phosphatase Cdc25 at Ser287 (of Xenopus Cdc25; Ser216 of human Cdc25C), a site that suppresses Cdc25 during interphase. Moreover, PP1 recognizes Cdc25 directly by interacting with a PP1-binding motif in the Cdc25 N-terminus. We have also found that 14-3-3 binding to phospho-Ser287 protects Cdc25 from premature dephosphorylation. Upon entry into M phase, 14-3-3 removal from Cdc25 precedes Ser287 dephosphorylation, suggesting the existence of a phosphatase- independent pathway for 14-3-3 removal from Cdc25. We show here that this dissociation of 14-3-3 from Cdc25 requires the activity of the cyclin-dependent kinase Cdk2, providing a molecular explanation for the previously reported requirement for Cdk2 in promoting mitotic entry. Collectively, our data clarify several steps important for Cdc25 activation and provide new insight into the role of PP1 in Cdc2 activation and mitotic entry.     

A neuromusculoskeletal tracking method for estimating individual muscle forces in human movement

A neuromusculoskeletal tracking (NMT) method was developed to estimate muscle forces from observed motion data. The NMT method combines skeletal motion tracking and optimal neuromuscular tracking to produce forward simulations of human movement quickly and accurately. The skeletal motion tracker calculates the joint torques needed to actuate a skeletal model and track observed segment angles and ground forces in a forward simulation of the motor task. The optimal neuromuscular tracker resolves the muscle redundancy problem dynamically and finds the muscle excitations (and muscle forces) needed to produce the joint torques calculated by the skeletal motion tracker. To evaluate the accuracy of the NMT method, kinematics and ground forces obtained from an optimal control (parameter optimization) solution for maximum-height jumping were contaminated with both random and systematic noise. These data served as input observations to the NMT method as well as an inverse dynamics analysis. The NMT solution was compared to the input observations, the original optimal solution, and a simulation driven by the inverse dynamics torques. The results show that, in contrast to inverse dynamics, the NMT method is able to produce an accurate forward simulation consistent with the optimal control solution. The NMT method also requires 3 orders-of-magnitude less CPU time than parameter optimization. The speed and accuracy of the NMT method make it a promising new tool for estimating muscle forces using experimentally obtained kinematics and ground force data.