Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxo-pregnanes

The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5α-pregnane-3β-ol-20-one 3HS:BSA (5α-20-one EIA) and 5β-pregnane-3α-ol-20-one 3HS:BSA (5β-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1–3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5α-pregnane-3, 20-dione, 5α-pregnane-3α-ol-20-one, and 5α-pregnane-3β-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3–7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 μg/g) by week 11. During months 6–13 concentrations in the 5α-20-one and in the 5β-20-one EIA (5–11 μg/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5–3 μg/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody. © 1996 Wiley-Liss, Inc.     

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa

BackgroundThe awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent.MethodologyWe searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients.FindingsA total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections.ConclusionThe small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies.     

Early kinetics of calprotectin in plasma following inguinal hernia surgery

Calprotectin is one of the most abundant proteins of neutrophil granulocytes. It is released upon neutrophil activation and is considered a sensitive and clinically useful marker for neutrophil-mediated inflammation, including bacterial infections. However, early kinetics of calprotectin activation following inflammatory activation has hitherto been unknown. The aim of the present study was to determine the early phase of the kinetics of calprotectin, in comparison with the inflammatory markers CRP, IL-6, TNF-α, and procalcitonin, in plasma following a standardized temporary mild inflammatory response, using uncomplicated inguinal hernia surgery as a model. The study cohort consisted of 17 adult patients (15 male and 2 female) undergoing elective surgery for hernia. Values of calprotectin increased significantly at 2 h following surgery, and continued to increase to reach the highest level at 24–36 h after surgery, values still not exceeding upper normal reference level. This contrasts to IL-6 and CRP, for which an elevation was found first later, 4 h and 24–36 h post-surgery, respectively, for IL-6, and CRP. No significant increase was seen for TNF-α, or procalcitonin. The data demonstrate a very rapid and significant but modest increase in calprotectin following induction of mild inflammation, supporting that calprotectin can be useful for early detection of inflammatory response.     

Placental failure and impaired vasculogenesis result in embryonic lethality for neuropathy target esterase-deficient mice

Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE(+/-) mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.     

Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.     

Craniofacial anthropometric pattern profile in hypohidrotic ectodermal dysplasia--application in detection of gene carriers

Hypohidrotic ectodermal dysplasia (HED) is characterized by clinical manifestations of severe hypodontia or anodontia, hypotrichosis, hypohidrosis, and specific facial appearance. Affected males show complete expression of clinical features of this condition. Their mothers, who are gene carriers, express only some signs, which are usually very mild. Currently available clinical methods are not sufficient for routine identification of the HED heterozygous gene carriers. The purpose of this study was to identify and describe the facial characteristics of HED patients and their mothers and to evaluate the usefulness of craniofacial pattern profile analysis (CFPP) in the diagnosis of this syndrome and the detection of gene carriers. In this study six affected males and their mothers were evaluated. Z-scores for each variable were calculated and compared with age- and sex-matched controls. Anthropometric analysis showed a specific dysmorphic pattern in CST patients that includes decreased skull base width (t-t: -1.67 Z); decreased forehead width (ft-ft: -1.8 Z), decreased midface depth (sn-t: -2.02 Z), markedly decreased total facial height (n-gn: -3.4 Z), and markedly decreased maxillary arc (t-sn-t: -2.5 Z). Gene carriers showed a similar tendency in their pattern profiles. They showed the same tendency towards lower Z-values for forehead width, facial height, and mouth width. The values for these measurements were between those of the affected and healthy controls. The most pronounced findings were increased head width (eu-eu: +2.83 Z), increased lower face width (go-go: +2.06 Z), and reduction of total facial height (n-gn: -0.95 Z). They also displayed increased nose width (al-al: +2.41 Z) and increased biocular distance (ex-ex: +2.01 Z). When used in conjunction with other methods the anthropometrics pattern profile analysis can considerably enhance detection of gene carriers for HED and increase objective assessment of the craniofacial region in HED patients.