Molecular cloning of the uteroferrin-associated protein, a major progesterone-induced serpin secreted by the porcine uterus, and the expression of its mRNA during pregnancy

The uteroferrin(Uf)-associated basic proteins (UfAP) are a group of three (Mr = 42K, 48K, and 50K) antigenically related, basic glycoproteins secreted by the porcine uterus under the influence of progesterone (P4) which exist as heterodimers (Mr = 80,000) with the iron-binding acid phosphatase, Uf. Several UfAP cDNA clones from a day-60 pregnant pig uterine endometrial cDNA library have been cloned and sequenced. The UfAP mRNA is approximately 1400 bases long and has a single open reading frame of 1251 bases with two start codons at positions 64 and 79 from the 5'-end. UfAP mRNA content of the endometrium increases as pregnancy proceeds, reaching maximum levels around day 70 and then remaining relatively constant in late gestation (days 70 to 110). The pro-form of the UfAP minus signal sequence appears to be 392 amino acids in length and has four potential N-linked glycosylation sites Asn107, Asn197, Asn243, and Asn315. Comparison of the NH2-terminal sequences of the individual UfAP poly-peptides with the amino acid sequence deduced from the cDNA has indicated a series of at least four posttranslational proteolytic processing steps which generate the various molecular forms of the UfAP. The deduced amino acid sequence of UfAP shares considerable identity with several protease inhibitors and hormone-binding proteins that are members of the serpin superfamily of proteins. The UfAP amino acid sequence also exhibits about 55% sequence identity with the P4-induced uterine milk proteins (UTMP) of the sheep. Since the UfAP and UTMP share many biosynthetic and structural features that include site of biosynthesis in the endometrium, P4-responsiveness, the presence of the mannose 6-phosphate lysosomal recognition marker, and considerable sequence similarity, the UfAP and the UTMP may have homologous function which for both still remains obscure.     

Production of leukotrienes and prostaglandins in the rat uterus during peri-implantation period

We have measured by radioimmunoassay the production of leukotrienes (LTC4 and LTB4) and prostaglandins (PGE2 and PGF2 alpha) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC4 or LTB4 remained unaltered on days 1-3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE2 and PGF2 alpha showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: vasodilators and agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation per se.     

Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding to bovine trophoblast protein-1: a comparison with ovine trophoblast protein-1 and bovine interferon-alpha II

Bovine trophoblast protein-1 (bTP-1) is a secreted glycoprotein that consists of several forms differing slightly in mol wt and isoelectric point. It is produced by bovine conceptuses after about day 15 of pregnancy and is believed to play a key role in signalling the presence of an embryo to the mother. In this study, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18-19 bovine conceptus poly(A)+ mRNA. Base sequencing of several cDNAs indicated that multiple mRNAs for bTP-1 exist. Northern blotting and primer extension experiments showed that the mRNAs average about 1 kilobase in length. One apparently full-length cDNA clone consisted of 1035 bases up to the beginning of the poly(A) tail. It contained an open reading frame of 195 codons which began at a position 79 bases from the 5' end. Its entire sequence was 85% identical to that of a cDNA for the immunologically related ovine trophoblast protein-1 (oTP-1) and about 79% identical to that for a bovine interferon-alpha II (IFN alpha II). The highest conservation of sequence (greater than 90%) was noted in the 3'-untranslated sequences of the bTP-1 and oTP-1 cDNAs. The deduced amino acid sequence of bTP-1 shared 80% identity with oTP-1, between 45-55% with human, rodent, porcine, and bovine IFNs of the alpha 1 subfamily and about 70% with a bovine IFN alpha II. A single potential site for N-glycosylation was noted at Asn78. These results show that bTP-1, like its ovine counterpart oTP-1, is structurally related to the IFN alpha S. We suggest that these embryonic IFNs play a role in controlling immunoreactions at the trophoblast-uterus interface as well as triggering other maternal responses to pregnancy.     

Regulation of glutamine synthetase I fromRhizobium meliloti

Glutamine synthetase I fromRhizobium meliloti was found to be inhibited by adenosine 5-monophosphate, alanine, glycine, carbamyl phosphate, cytidine 5-triphosphate, tryptophan, histidine, and glucosamine-6-phosphate. Each inhibitor was independent in its action and the effect was cumulative when more than one inhibitor was added.     

Configuration of PKCα-C2 Domain Bound to Mixed SOPC/SOPS Lipid Monolayers

X-ray reflectivity measurements are used to determine the configuration of the C2 domain of protein kinase Cα (PKCα-C2) bound to a lipid monolayer of a 7:3 mixture of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of PKCα-C2 and a slab model representation of the lipid layer. The configuration of lipid-bound PKCα-C2 is described by two angles that define its orientation, θ = 35° ± 10° and φ =210° ± 30°, and a penetration depth (=7.5 ± 2 Å) into the lipid layer. In this structure, the β-sheets of PKCα-C2 are nearly perpendicular to the lipid layer and the domain penetrates into the headgroup region of the lipid layer, but not into the tailgroup region. This configuration of PKCα-C2 determined by our x-ray reflectivity is consistent with many previous findings, particularly mutational studies, and also provides what we believe is new molecular insight into the mechanism of PKCα enzyme activation. Our analysis method, which allows us to test all possible protein orientations, shows that our data cannot be explained by a protein that is orientated parallel to the membrane, as suggested by earlier work.     

Fish Gut Microbiome: Current Approaches and Future Perspectives

In recent years, investigations of microbial flora associated with fish gut have deepened our knowledge of the complex interactions occurring between microbes and host fish. The gut microbiome not only reinforces the digestive and immune systems in fish but is itself shaped by several host-associated factors. Unfortunately, in the past, majority of studies have focused upon the structure of fish gut microbiome providing little knowledge of effects of these factors distinctively and the immense functional potential of the gut microbiome. In this review, we have highlighted the recently gained insights into the diversity and functions of the fish gut microbiome. We have also delved on the current approaches that are being employed to study the fish gut microbiome with an aim to collate all the knowledge gained and make accurate conclusions for their application based perspectives. The literature reviewed indicated that the future research should shift towards functional microbiomics to improve the maximum sustainable yield in aquaculture.