The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): III. Detection of IgA antibodies in human milk that bind to bacterial toxins implicated in SIDS

Two toxin-producing bacteria implicated in sudden infant death syndrome (SIDS) are Staphylococcus aureus and Clostridium perfringens. Epidemiological studies have shown that breast feeding reduces an infant's risk of SIDS. This protective effect could be due partly to IgA antibodies to these toxins in human milk. The aim of this work was to use a quantitative ELISA to determine levels of IgA antibodies that bound to toxic shock syndrome toxin (TSST-1), staphylococcal enterotoxin C (SEC) and C. perfringens enterotoxin A (CEA) in individual samples of human milk. All samples of milk tested contained IgA antibodies that bound to the bacterial toxins. For individual samples, IgA bound to TSST-1, SEC and CEA were in the range of 900-3100 ng ml(-1), 1000-3600 ng ml(-1) and 1000-4300 ng ml(-1) respectively. Isolation of S. aureus from mothers donating breast milk samples was used to determine if the presence of bacteria affected IgA levels which bound TSST-1 and SEC. For 3/5 samples with levels above the upper limit of the standard deviation (2375 ng ml(-1)) for IgA bound to TSST-1, S. aureus was isolated from the mother whilst 4/5 samples found to contain levels above the upper limit of the standard deviation (2627 ng ml(-1)) for IgA bound to SEC, had S. aureus isolated from the mother. In conclusion, if bacterial toxins do play a role in precipitating a SIDS death, the presence of IgA antibodies to toxins in breast milk, but not in infant formula, might contribute to the protective effect of breast feeding in relation to SIDS.     

Small genes/gene-products in Escherichia coli K-12

Forty-two protein spots of observed M_r 6–15 kDa were resolved by two-dimensional gel electrophoresis, stained by Coomassie blue and subjected to Edman microsequencing. All of the proteins could be related back to their encoding open reading frames, thereby vindicating the bioinformatic tools currently utilised in their identification. However, only 14/42 gene-products were expressed as annotated. Translation was confirmed for 14 open reading frames with no attributed function (EcoGene Y-entries), while N-terminal sequence allowed the start codon to be accurately annotated for the genes yjgF, yccU, yqiC, ynfD, and yeeX. The methionine start codon was cleaved in 11 gene-products (AtpE, Hns, RpoZ, RplL, CspC, YccJ, YggX, YjgF, HimA, InfA, RpsQ) and a further five showed loss of a signal peptide (PspE, HdeB, HdeA, YnfD, YkfE). Internal (Tig, AtpA, TufA) and N-terminal fragmentation (CspD, RpsF, AtcU) of much larger proteins was also detected, which may have resulted from physiological or translational processes. M_r and pI isoforms were detected respectively for PtsH and GatB, each being phosphoproteins, as well as RplY which manifested differences with respect to predicted M_r and pI. In addition, YjgF was shown to belong to a small gene family of unknown function with ancient conserved regions across procaryotes and eucaryotes. YgiN was revealed to have a paralogue and orthologues in Bacillus subtilis, Synechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoea, and Rhodococcus erythropolis. Orthologues are also reported for YihD, YccU and YeeX. Of the 14 Y-genes, only YkfE possessed no detectable orthologues. These results highlight the need to complement genomic analysis with detailed proteomics in order to gain a better understanding of cellular molecular biology, while the confirmation of the open reading frame start codon using Edman degradation protein microsequencing has yet to be superseded by recent advances in mass spectrometry.     

Influence of thrombin on proliferation and prostaglandin production in cultured bovine endometrial cells

The control of cell proliferation by thrombin was studied in vitro in cultured epithelial and stromal cells of the endometrium. The effect of thrombin was studied after chronic treatment (72 hr) in medium containing 10% fetal bovine serum (FBS) combined or not with sex steroids. Thrombin inhibited slightly the proliferation (based on DNA measurements) only in epithelial cells (P < 0.05). 17β-estradiol (E) and progesterone (P4) had no mitogenic effects. The presence of functional thrombin receptors was estimated by stimulation of second messenger generation in response to increasing doses of thrombin (0-1,500 ng/ml). In confluent cultures of epithelial cells, the addition of thrombin for 10 min stimulated cAMP production by 50% with a maximal response at 500 ng/ml (P < 0.05). Similarly, in stromal cells, thrombin stimulated cAMP production in a dose-dependent manner (P < 0.01). Generation of inositol-phosphates was also stimulated by 50% in epithelial cells (P < 0.03), with a maximal response at 500 ng/ml, and by 45% in stromal cells (P < 0.01), with a maximal response at 50 ng/ml. The effect of thrombin on cell proliferation was investigated by ³H-thymidine incorporation in serum-free medium for 24 hr. Thrombin inhibited incorporation in epithelial cells (P < 0.0001) in a dose-dependent manner. Conversely, thrombin stimulated significantly incorporation of stromal cells (P < 0.05) at 50 ng/ml. The effect of sex steroids was also evaluated and it was found that E had no effect on cell proliferation, while P4 inhibited the incorporation in both epithelial (P < 0.001) and stromal cells (P < 0.001). The effect of a combined treatment with thrombin and E inhibited both epithelial (P < 0.001) and stromal cell (P < 0.001) growth, but a combination of thrombin and P4 had no additional effect on growth compared to P4 alone. Further investigation of the role of thrombin has been carried out by measuring prostaglandin (PG) responses. Addition of thrombin for 24 hr inhibited PGF_2α production by epithelial cells (P < 0.0001) but had no effect on PGE₂ production by stromal cells. Therefore, functional receptors for thrombin appear to be present in epithelial and stromal cells of the bovine endometrium. The minimal effect of thrombin alone or in combination with sex steroids on endometrial cell proliferation in vitro combined with the evidence of functional thrombin receptor in these cells, suggest that: (1) the effect of sex steroids in cultured endometrial cells is not modulated by the presence of thrombin, and (2) other factors are necessary for the full expression of mitogenic responses to sex steroids in vitro. © 1996 Wiley-Liss, Inc.     

Degenerative Osteoarthropathy in Laboratory Housed Xenopus (Silurana) tropicalis

In this case study, 15 adult laboratory Xenopus (Silurana) tropicalis (7 adult males and 8 adult females) were examined for nodular enlargements of the clawed digits (digits 0, I, II, and III) on the hind feet. Radiographs showed smoothly margined, rounded, peripherally mineralized lesions arising from the distal phalanges of digits 0-III with osteoproductive and osteolytic components in all frogs. Micro computed tomography (microCT) scans further revealed interphalangeal (IP), metacarpophalangeal (MCP), and metatarsophalangeal (MTP) joint osteoarthritis characterized by periarticular new bone formation, rounded mineral foci both peripherally and centrally within the joints, and more rarely, linear mineralization palmar/plantar to the joints in the flexor tendons. In the nonclawed digits, the shape of the distal phalanx was variably distorted and both subluxation and malangulation of IP joints were identified. Histologically, nodules corresponded to a peripheral rim of mature cortical bone surrounding central adipose tissue, scattered hematopoietic elements, and residual bone of the distal phalanx. Occasionally, the peripheral rim of cortical bone extended proximally to encompass the distal aspect of adjacent phalanx. MCP, MTP and IP joint spaces of most digits exhibited widespread osteoarthritis characterized by periarticular cartilaginous or osseous metaplasia, bony remodeling, and less frequently, granulomatous osteomyelitis. Nutritional analyses of the feed did not indicate imbalances nor were the lesions consistent with metabolic bone disease. The exact etiopathogenesis of these lesions is unknown; however, we hypothesize that the osteoarthritic changes are due to a combination of the frogs’ mature age, the unique structure of the Xenopus spp. claw, genetics and biomechanical forces on the digits and distal phalanges of the hind feet.

Xenopus spp. are a well-established model in biomedical research and are commonly used in the fields of embryology and vertebrate developmental biology, endocrinology, toxicology, cancer, and genomics.^{1-3,5,10,11,13,16,17,19,20,22} In research laboratory environments, X. tropicalis are typically housed in either static or recirculating aquatic systems in water maintained at 25 to 28 °C, with a pH between 6.8 and 8.5, and a hardness between 100 and 300 mg/L CaCo₃.⁷ Housing tanks range in size from 1 L or more, depending on the needs of the laboratory and the type and configuration of the housing system. Stocking density ranges from approximately 1-5 adult frogs/L per tank.⁷ Laboratory X. tropicalis are commonly fed commercially available complete and balanced pelleted diets 3 times per week, or as appropriate for their different life stages.^7,18,21 With stringent husbandry and disease surveillance protocols in place, laboratory-reared and housed Xenopus spp. generally remain healthy well through adulthood and their prime egg-producing years and can live 10 y or longer in captivity.⁷To date, most reports describing disease in laboratory or wild Xenopus spp. are attributed to infectious agents (bacterial, viral, or parasitic) and/or conditions related to water quality aberrations or pollutants.⁷ Naturally occurring bone diseases due to nutritional appear to be rare in laboratory Xenopus spp., although they have been described in other frogs and amphibians.^6,12,23 Reports of other spontaneous skeletal diseases in laboratory Xenopus are particularly rare. One group²⁵ recently described axial skeletal deformities in genetically engineered and wild type laboratory Xenopus spp. Here we report spontaneously occurring appendicular skeletal degenerative osteoarthropathy with hindlimb distal phalangeal nodules (digits 0-III) in mature, laboratory X. tropicalis, as confirmed by radiography, computed tomography and histopathology.     

Viral RNA gymnastics

Retroviruses have a stretch of RNA that dimerizes during viral particle formation. A new study suggests that RNA flexibility in the monomeric form may facilitate dimerization or other RNA-dependent viral functions.     

Involvement of two Hox genes and Otx in echinoderm body-plan morphogenesis in the sea urchin Holopneustes purpurescens

The expression of Hox11/13 and Hox5 orthologues in the adult echinoid rudiment in the vestibula larva of Holopneustes purpurescens is described from whole mounts and sections of whole mounts after mRNA in situ hybridization. The Hox5 orthologue is HpHox5, which was isolated here. The expression of HpHox11/13 in the epithelium of the vestibule is aboral to the expression of HpHox5. HpHox5 is expressed in the epithelium of the vestibule floor where the secondary podia develop. The expression of HpHox11/13 and HpHox5 contrasts with the expression of an Otx orthologue, HprOtx, in the circum-oral nerve ring, the radial nerves and the neuroepithelium around the bases of the primary podia. From the expression patterns, we conclude that the two Hox genes are involved in the growth of a metameric series of secondary podia from a growth zone aboral to each primary podium, with the older podia nearer the circum-oral nerve ring. With respect to echinoderm body-plan polarities, we conclude that the growth zone is posterior relative to the anterior circum-oral nerve ring. The metamerism generated in this echinoderm from a posterior growth zone thus might not be generated differently from the way it is generated in bilateral animals.     

Two-dimensional liquid chromatography/tandem mass spectrometry analysis of Gradiflow fractionated native human plasma

One of the major challenges facing protein analysis is the dynamic range of protein expression within massively complex samples (Corthals, G. L. et al.., Electrophoresis 2000, 21, 1104-1115). In plasma this difference is as great as ten orders of magnitude, and this is currently beyond the range of detection achievable by any of the analytical techniques. Plasma has the additional challenge of having a few highly abundant proteins, such as albumin, which mask the detection of lower abundance and biologically significant proteins. The use of the Gradiflow BF400 as a fractionation tool to deplete highly abundant albumin from human plasma is reported here. A sequential three-step protocol was performed on five plasma samples as part of the International Plasma Proteome Project organised by the HUPO; four containing different anticoagulants: EDTA, citrate, heparin and a control sample (NIBSC); and a serum sample. Plasma from an alternate source also underwent fractionation and served as an in-house control. Time modulation between 1 and 7 h was observed for the depletion of albumin from these samples. Following albumin depletion, each fraction was trypsin-digested and the peptides were fractionated further using a 2-D LC-MS/MS. Differences in the total number of proteins identified for each sample were also noted.     

MS characterization of multiple forms of alpha-amylase in human saliva

Alpha-amylase is a major and well-characterized component of human saliva. Recent proteomic studies suggested that this protein could be observed in more than twenty spots on 2-D gels of salivary proteins. The aim of this work was to investigate this unexpected redundancy. 2-D gel electrophoresis was combined with systematic MALDI-TOF MS analysis. More than 140 protein spots identifying the alpha-amylase were shown to constitute a stable but very complex pattern. Careful analysis of mass spectra and simultaneous hierarchical clustering of the observed peptides and of the electrophoretic features of spots allowed one to define three major groups. A main class grouping 90 spots was shown to correspond to full length alpha-amylases that can be assumed to include isoforms and post-translationally modified forms, a subset of this class being demonstrated to be N-glycosylated. A second group included short alpha-amylases that are differently truncated in a non-random manner, very likely in the oral cavity. The last class grouped alpha-amylase forms showing both the N- and C-terminal sequences of the enzyme but displaying a molecular weight that was up to 50% lower than that of the native protein. It is speculated that the last group of alpha-amylase spots could correspond to proteins submitted to internal deletions prior to the secretion.     

Digit ratio varies with sex, egg order and strength of mate preference in zebra finches

The steroid environment encountered by developing vertebrates has important organizational effects on physiology and behaviour that persist throughout an organism's lifetime. Optimal allocation of maternal steroids to zygotes may be difficult to achieve because of the sexually antagonistic effects of steroids; thus, for example, a hormone environment beneficial to a developing male may be much less beneficial to a developing female. Research into the important topic of how mothers might adaptively adjust steroid titres experienced by particular young has been constrained by the difficulty of measuring the steroid environment experienced by the embryo at critical times in development. A potential approach to this problem has been suggested by research on variation in digit ratios in humans, where the ratio of the length of the second and fourth digits reflects the steroid environment experienced by the foetus; notably, digit 4 lengthens in response to androgens. In light of the conservative nature of homeobox genes regulating early development in tetrapods, we questioned whether a sex difference in digit ratio exists in a passerine bird, the zebra finch, Taeniopygia guttata castanotis, and whether observed variation in the ratio is consistent with the previously reported pattern that androgen allocation to zebra finch egg yolk declines across laying order. We established an aviary population of outbred, wild-type zebra finches, and allowed them to breed freely. Hatchlings were marked to correspond to their egg order, and their digit ratios were measured after birds reached adulthood. We found that digit ratio increased across egg order, which is consistent with a pattern of decreasing androgen allocation. Moreover, digit ratios differed between the sexes. We also investigated whether variation in digit ratio among adult females predicted variation in their performance in mate-choice tests. Digit ratio accounted for almost 50% of the variance in strength of female preference for an attractive male trait: specifically, females with higher (presumably less 'androgenized') ratios had stronger preferences for attractive males. Digit ratio may prove to be an extremely useful tool for addressing a wide range of questions about vertebrate differentiation and behaviour.