Purification and characterization of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor.

The mRNA coding for the common precursor of corticotropin and beta-lipotropin has been purified to homogeneity from neurointermediate lobes of bovine pituitaries. The homogeneity of the mRNA preparation is evidenced by analysis of its translation product, electrophoresis on polyacrylamide gel in the presence of formamide and analysis of the kinetics of hybridization with its cDNA. The purification procedure involves the isolation of RNA from membrane-bound polysomes, chromatography on oligo(dT)-cellulose and on poly(U)-Sepharose and sucrose density gradient centrifugation. The mRNA has a molecular weight of approximately 450000, equivalent to approximately 1360 nucleotides in length, and contains a polyadenylate sequence with an average length of 68 nucleotides. The size of the mRNA is sufficiently large to encode the corticotropin/beta-lipotropin precursor.     

Enhanced synthesis of type IV collagen in cultured arterial smooth muscle cells associated with phenotypic modulation by dimethyl sulfoxide

The effect of dimethyl sulfoxide (DMSO) on synthesis of basement membrane collagen in cultured smooth muscle cells was evaluated. DMSO promoted phenotypic modulation of cells from the synthetic state to the contractile state accompanied by formation of basement membranes. By immunofluorescence using monospecific antibody against type IV collagen, type IV collagen was identified not only in the cell cytoplasms but intensely along the cell surfaces in the cultures treated with DMSO for 7 days, as compared with untreated cultures. Electron microscopic immunohistochemistry revealed the presence of type IV collagen both in the basement membrane region and in the rough endoplasmic reticulum of DMSO-treated cells. Such an enhancement of type IV collagen synthesis appears to be expressed as a result of the phenotypic changes of smooth muscle cells to the contractile state modulated by DMSO.     

Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina.

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.     

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F₁-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F₁-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F₁-ATPase from Bacillus subtilis (BF₁), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF₁. We conclude that the ε subunit relieves BF₁ from MgADP inhibition.     

Relevance of Distinct Monocyte Subsets to Clinical Course of Ischemic Stroke Patients

Background and Purpose

The most common strategy for treating patients with acute ischemic stroke is thrombolytic therapy, though only a few patients receive benefits because of the narrow time window. Inflammation occurring in the central nervous system (CNS) in association with ischemia is caused by immune cells including monocytes and involved in lesion expansion. If the specific roles of monocyte subsets in stroke can be revealed, they may become an effective target for new treatment strategies.

Methods

We performed immunological examinations of 36 consecutive ischemic stroke patients within 2 days of onset and compared the results with 24 age-matched patients with degenerative disorders. The stroke patients were repeatedly tested for the proportions of monocyte subsets in blood, and serum levels of pro- and anti-inflammatory cytokines immediately after admission, on days 3-7 and 12-16 after stroke onset, and on the day of discharge. In addition, immunological measurements were analyzed for relationships to stroke subtypes and complications, including progressive infarction (PI) and stroke-associated infection (SAI).

Results

Monocyte count was significantly increased from 0–16 days after stroke as compared to the controls (p<0.05). CD14^highCD16^- classical and CD14^highCD16⁺ intermediate monocytes were significantly increased from 0-7 and 3-16 days after stroke, respectively (p<0.05), whereas CD14 ^dimCD16^high non-classical monocytes were decreased from 0–7 days (p<0.05). Cardioembolic infarction was associated with a persistent increase in intermediate monocytes. Furthermore, intermediate monocytes were significantly increased in patients with PI (p<0.05), while non-classical monocytes were decreased in those with SAI (p<0.05). IL-17A levels were positively correlated with monocyte count (r=0.485, p=0.012) as well as the percentage of non-classical monocytes (r=0.423, p=0.028), and negatively with that of classical monocytes (r=-0.51, p=0.007) during days 12-16.

Conclusions

Our findings suggest that CD14^highCD16⁺ intermediate monocytes have a role in CNS tissue damage during acute and subacute phases in ischemic stroke especially in relation to cardioembolism.