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H Hemilä

Publications and source records attributed to H Hemilä.

At least 37 records · Page 2Linked to original sources
PubMed1992

Improving the production of E. coli beta-lactamase in Bacillus subtilis: the effect of glucose, pH and temperature on the production level.

Bacillus subtilis has been considered a promising host for the production of foreign proteins. However, proteases released by the host organism can often cause rapid breakdown of secreted heterologous proteins. Here we report that the addition of 6% glucose and 100 mM potassium phosphate to the growth medium significantly reduces the degradation of E. coli TEM beta-lactamase secreted from B. subtilis, when applying an expression system based on B. amyloliquefaciens alpha-amylase. The yield of beta-lactamase was increased 10-20-fold when compared to the yield in Luria medium. The promoter of B. amyloliquefaciens alpha-amylase gene is repressed by glucose. However, here we show that the repression does not take place in a multicopy plasmid, thus enabling our approach to efficiently reduce the protease action by catabolite repression. We have also studied the role of pH and temperature on the beta-lactamase production in laboratory scale bioreactors. Low temperature and low pH are both favorable for a high level beta-lactamase production by the high copy plasmid construction.

Bacillus subtilis↗
PubMed1992

Vitamin C and the common cold.

The effect of vitamin C on the common cold has been the subject of several studies. These studies do not support a considerable decrease in the incidence of the common cold with supplemental vitamin C. However, vitamin C has consistently decreased the duration of cold episodes and the severity of symptoms. The benefits that have been observed in different studies show a large variation and, therefore, the clinical significance may not be clearly inferred from them. The biochemical explanation for the benefits may be based on the antioxidant property of vitamin C. In an infection, phagocytic leucocytes become activated and they produce oxidizing compounds which are released from the cell. By reacting with these oxidants, vitamin C may decrease the inflammatory effects caused by them. Scurvy, which is caused by a deficiency in vitamin C, is mostly attributed to the decreased synthesis of collagen. However, vitamin C also participates in several other reactions, such as the destruction of oxidizing substances. The common cold studies indicate that the amounts of vitamin C which safely protect from scurvy may still be too low to provide an efficient rate for other reactions, possibly antioxidant in nature, in infected people.

Antioxidants↗
PubMed1991

Lipoamide dehydrogenase of Staphylococcus aureus: nucleotide sequence and sequence analysis.

A complex of four proteins was previously isolated from Staphylococcus aureus. The complex had a strong interaction with membrane bound ribosomes, which suggested that it may be involved in protein secretion. However, the complex was identified as pyruvate dehydrogenase (PDH), which disproved the direct role of the complex in protein secretion. Here we report the nucleotide sequence of the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, which encodes lipoamide dehydrogenase (LPD). The pdhD gene encodes a protein of 468 amino acids, with a molecular mass of 49.5 kDa. The protein is closely related to other lipoamide dehydrogenases from bacteria and eukaryotes. The possible role of membrane bound lipoamide dehydrogenase is briefly discussed.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins.

Sec Y is an integral membrane protein which participates in the translocation of proteins through the bacterial cell membrane. We have cloned the sec Y gene of Lactococcus lactis, and found its deduced protein sequence, 439 amino acids long, to be similar in length to the previously determined Sec Y proteins of Escherichia coli, Bacillus subtilis and Mycoplasma capricolum. Comparison of the L. lactis Sec Y to the 3 other Sec Y proteins revealed 90 conserved amino acid residues (21%). Nearly half of the conserved residues are clustered in 2 of the 10 transmembrane segments, and in 2 of the 6 cytoplasmic regions. Some of the conserved regions are apparently responsible for the interactions of Sec Y with signal sequences, and the proteins SecE and SecA.

Amino Acid Sequence↗
PubMed1991

Sequence of a PAL-related lipoprotein from Bacillus subtilis.

The sequence of a small Bacillus subtilis lipoprotein is reported. The gene encodes a protein of 124 amino acids, which shows a low but statistically significant homology to the peptidoglycan-associated lipoproteins (PAL) of Escherichia coli and Haemophilus influenzae. Although the functions of these proteins have not been confirmed, they are obviously structural proteins. In E. coli the gene for the peptidoglycan-associated lipoprotein appears to be essential.

Amino Acid Sequence↗
PubMed1991

Production of heterologous proteins in Bacillus subtilis: the effect of the joint between signal sequence and mature protein on yield.

We have previously made a set of DNA constructs by fusing the mature part of Bacillus licheniformis alpha-amylase with the signal sequence of B. amyloliquefaciens alpha-amylase at various distances from the signal sequence cleavage site. We observed that the level of alpha-amylase production in B. subtilis depended strongly on the distance of the junction from the signal sequence cleavage site, with quite a sharp optimum distance. To test whether the effect is limited to the pair of alpha-amylase signal sequence and mature protein, we analysed the protein production in a set of constructs in which an Escherichia coli beta-lactamase was similarly joined at different distances from the alpha-amylase signal sequence. Also in this case the distance seemed to be an important factor in affecting the level of production in B. subtilis. The observed effect might depend on the modulation of pre-protein folding, which in turn could affect the secretion level.

Amino Acid Sequence↗
PubMed1991

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis.

The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-poly-acrylamide gel electrophoresis characterizations. The NH2-terminal sequence analysis of the secreted PME revealed two different NH2-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and alpha-amylase signal peptide.

Bacillus subtilis↗
PubMed1991

Nucleotide sequence of a Lactococcus lactis gene cluster encoding adenylate kinase, initiation factor 1 and ribosomal proteins.

We have previously isolated a putative promoter from the Lactococcus lactis subsp. lactis chromosome. We now report the sequence of the promoter fragment and its extension in the 5'-direction. The region contains several open-reading frames which correspond to ribosomal protein L15, SecY, adenylate kinase, initiation factor 1 and ribosomal proteins B and S13. The order of the genes, rplO (L15), secY, adk, infA, rpmJ (B) and rpsM (S13), is similar to that in the spc and alpha operon region of Bacillus subtilis, with the exception of the map gene, coding for methionine amino peptidase, which is located between adk and infA in B. subtilis. The putative promoter is located between adk and infA.

Adenylate Kinase↗
PubMed1990

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase.

We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion.

Amino Acid Sequence↗
PubMed1989

Production of diphtheria toxin CRM228 in B. subtilis.

The gene coding for a nontoxic diphtheria toxin (DT), tox228, was isolated from lysogenic Corynebacterium diphtheriae and cloned into pBR322. A mature form of the tox228 gene, lacking its signal sequence, was expressed in Bacillus subtilis using a B. amyloliquefaciens alpha-amylase secretion vector. To test the possibility of producing partially deleted DT molecules, which could be used for cell-directed toxin conjugates, a truncated form lacking 151 amino acids from the C-terminus of the DT was generated by oligonucleotide mutagenesis. Both the truncated and intact DT were efficiently secreted into the culture medium. During prolonged cultivation, the truncated form was less stable than the intact DT molecule.

Amino Acid Sequence↗
PubMed1986

A re-evaluation of nutritional goals--not just deficiency counts.

There is considerable controversy about the soundness and relevance of so-called "megavitamin" therapy for various illnesses. In this article it is suggested that this disagreement is caused to a large extent by the use of two very different approaches to nutrition, which are referred to here as the "nutritional need" and "optimal intake" approaches. It is clear that a re-evaluation of the goals of nutrition is required, as nutritional recommendations deal mainly with the prevention of deficiency diseases and are not concerned with optimal levels of intake.

Ascorbic Acid↗
PubMed1985

Retinoids activate superoxide production by polymorphonuclear leucocytes.

Retinol and retinoic acid were effective activators of oxygen consumption by human polymorphonuclear leucocytes (PMN) in micromolar concentrations. In contrast, retinyl acetate was ineffective as an activator. Retinol caused activation only after a lag time, the length of which depended on retinol concentration. Oxygen consumption was due to superoxide production by PMN. Superoxide production was observed as superoxide dismutase-inhibitable cytochrome c reduction. Previously, retinoids have been reported to inhibit PMN activation by phorbol myristate acetate, a tumour promoter. This retinoid-induced inhibition of PMN activation has been suggested to be a mechanism by which retinoids may protect against carcinogenesis in animals. However, the retinoid concentrations at which PMN inhibition was reported were much higher than those found to cause activation in this study. We found that retinoic acid slightly inhibited phorbol myristate acetate-activated superoxide production, but only at concentrations that caused activation. In contrast, activation by formyl-Met-Leu-Phe was effectively inhibited at a retinoic acid concentration that did not cause activation by itself.

Diterpenes↗
PubMed1984

Activated polymorphonuclear leucocytes consume vitamin C.

Polymorphonuclear leucocytes (PMN) are known to produce superoxide and other oxygen derivatives upon activation as part of their microbicidal armory. Here we report that extracellular ascorbate is effectively oxidised by activated but not by resting human PMN in vitro. The oxidation of ascorbate is mainly caused by the superoxide that is generated by the activated cells, as shown by its effective inhibition by superoxide dismutase. However, myeloperoxidase, which may generate hypochlorite, also contributes to a significant extent. Ascorbate reduces superoxide to peroxide, as indicated by measurements of the stoichiometry of ascorbate and oxygen consumption. These results support the notion that extracellular ascorbate may serve as an important physiological protecting agent against oxygen radical damage in inflammation.

Ascorbic Acid↗