Temporal changes in serum prostaglandin F2alpha and oxytocin in dairy cows with short luteal phases after the first postpartum ovulation

Luteolysis in the cow depends upon an interaction between prostaglandin F(2alpha) (PGF(2alpha)) and oxytocin. The objectives of our study were 1) to determine oxytocin concentrations in postpartum dairy cows and 2) to identify the temporal relationship between oxytocin and PGF(2alpha) release patterns during luteolysis in normal and abbreviated estrous cycles in the postpartum period. Serum oxytocin and PGF(2alpha) metabolite (PGFM) concentrations from nine cows which had short estrous cycles (< 17 d) were compared with those of six cows which had normal estrous cycles. Serum basal oxytocin concentrations in short estrous cycle cows (23.7 to 31.1 pg/ml) were higher (P<0.05) than those of normal estrous cycle cows (14.6 to 19.8 pg/ml). Oxytocin concentrations increased to peak values in both short and normal cycle cows, during luteolysis. Basal PGFM concentrations (112.2 to 137.4 pg/ml) were higher in cows with short cycle (P<0.05) than in cows with normal cycles (62.9 to 87.5 pg/ml). The increase in PGFM concentrations during luteolysis was significant in both normal cycle and short cycle cows (P<0.05). Increases in serum PGFM concentrations were always associated with increases in serum oxytocin concentrations in normal cycle and short cycle cows and the levels decreased simultaneously before the subsequent estrus. Results support the idea of a positive relationship between PGF(2alpha) and oxytocin concentration during the estrous cycle as well as a possible synergistic action of these hormones in the induction of luteolysis in dairy cattle.     

Secretion of a1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Galβ1→4GLcNAc (Gal to Gal) a1,3 galactosyltransferase (a1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the a1,3GT activity. A recombinant and soluble form of the murine a1,3GT was produced in H293 cells (H293-a1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-a-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, a1,3GT and the time of incubation. The assay was also useful in monitoring a1,3GT activity during enzyme enrichment procedures. Using this assay, we found that a1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the a1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon. This revised version was published online in November 2006 with corrections to the Cover Date.     

Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.     

Location-dependent variations in the material properties of the anterior cruciate ligament.

Our recent anterior drawer studies in human cadaveric knees [Guan and Butler, Adv. Bioengng 17, 5 (1990); Guan et al., Trans. orthop. Res. Soc. 16, 589 (1991)] have suggested that anterior bundles of the anterior cruciate ligament (ACL) develop higher load-related material properties than posterior bundles. This was confirmed when we reevaluated the axial failure data for these bundle-bone specimens from an earlier study [Butler et al., J. Biomechanics 19, 425-432 (1986)]. The purpose of this study was to determine, in a larger data set, if anteromedial and anterolateral bundles of the anterior cruciate ligament exhibit significantly larger load-related material properties than the posterior ligament bundles. Seven ACL-bone units from seven donors (the three tissues from the original study plus four new ones) were subdivided into three subunits, preserving the bone insertions. The subunits were failed in tension at a constant strain rate (100% s-1) and four material properties were compared within and between donors. The anterior bundles developed significantly larger moduli, maximum stresses, and strain energy densities to maximum stress than the posterior subunits. Moduli for the anterior vs posterior subunits averaged 284 MPa vs 155 MPa, maximum stresses averaged 38 MPa vs 15 MPa, and strain energy densities averaged 2.7 N m cc-1 vs 1.1 N m cc-1, respectively. No significant differences were found, however, among strains to maximum stress or between any of the other properties for the two anterior subunits. These results are important to the design of ligament replacements and suggest new experiments designed to distinguish in vivo force levels in these ACL bands, a possible reason for the material differences.     

Phenotypic variation in Streptomyces sp. DSM 40537, a lipoteichoic acid producing actinomycete

Aims:  To reinvestigate the production of lipoteichoic acid (LTA) by the actinomycete strain Streptomyces sp. DSM 40537 (=ATCC 3351).
Methods and Results:  LTA was extracted and purified from strain Streptomyces sp. DSM 40537. The identification of the LTA was confirmed by Western blotting with a monoclonal antibody. During these studies, two stable phenotypic variants of DSM 40537 were obtained, one of which released a distinctive orange pigment. 16S rRNA gene sequencing of each variant yielded identical sequences and allowed phylogenetic analysis to be performed.
Conclusions:  Streptomyces sp. DSM 40537 was shown to exhibit stable morphological variation. The strain was confirmed to be a LTA-producing actinomycete and to belong to the Streptomyces albidoflavus cluster within the genus Streptomyces .
Significance and Impact of the Study:  These data provide important support for the hypothesis that the distribution of LTA is linked to that of wall teichoic acids and emphasizes the need to reinvestigate LTA distribution in actinomycetes.     

Bordetella pertussis, the causative agent of whooping cough, evolved from a distinct, human-associated lineage of B. bronchiseptica

Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu), and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica-like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu), and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed.     

Differential Expression of a Mycobacterium tuberculosis Protein Recognized by a Mouse Monoclonal Antibody

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Structure of the human gamma-fibrinogen gene. Alternate mRNA splicing near the 3' end of the gene produces gamma A and gamma B forms of gamma-fibrinogen

The gamma chain of human fibrinogen exists in 2 nonallelic forms, gamma A and gamma B, which differ only in their carboxyl termini. We have found that only one genomic locus exists for gamma-fibrinogen and that the gamma A and gamma B chains arise by alternate mRNA splicing near the 3' end of this gene. In contrast to the rat gamma B mRNA which is produced by alternate splicing with identical polyadenylation sites, human gamma B is produced when the eighth intervening sequence remains unspliced and a polyadenylation signal within this intron is used. The new carboxyl terminus is 16 amino acids longer than the gamma A protein, and although there is only minimal homology between the rat and human carboxyl termini they share a very high proportion of acidic amino acids.