New methods for solid phase peptide synthesis of transitionstate analog inhibitors of HIV-1 protease and DPP-IV

Summary A new and stereoselective method to synthesize hydroxyethylamine and hydroxymethylamide peptide bond isosteres is developed. The key step is the addition of 2-trimethylsilylthiazole to -aminoaldehydes, followed by transformation to -hydroxy--aminoaldehydes. The stereochemistry of the addition can be manipulated by the choice of the nitrogen substitution. The isosteres are easily synthesized via Solid Phase Peptide Synthesis, which rapidly gives the desired pseudopeptides.     

Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains

Lactic acid bacteria (LAB) have the enzyme potential to transform amino acids into aroma compounds that contribute greatly to cheese flavour. Generally, amino acid conversion by LAB is limited by their low production of -ketoglutarate since this -ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous -ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing -ketoglutarate from glutamate, greatly increased the conversion of amino acid to potent aroma compounds in cheese. Here we report the presence of glutamate dehydrogenase (GDH) activity required for the conversion of glutamate to -ketoglutarate in several natural LAB strains, commonly used in cheese manufacturing. Moreover, we show that the ability of LAB to produce aroma compounds from amino acids is closely related to their GDH activity. Therefore, GDH activity appears to be a major criterion for the selection of flavour-producing LAB strains, which could be used as a starter or as an adjunct to intensify flavour formation in some cheeses.     

Enhancement of vitamin E production in sunflower cell cultures

The most biologically active component of vitamin E, -tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms. Using sunflower cell cultures, a suitable in vitro production system of natural -tocopherol was established. The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol. Culture feeding experiments using biosynthetic precursors showed that -tocopherol production improved by 30% when homogentisic acid was used. Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in -tocopherol production when using cultures of longer subculture intervals. Compared to the starting plant tissue, an overall 100% increase of -tocopherol was reached by these sunflower cell cultures.     

A novel α-glucosidase produced by Bacillus amylolyticus

Bacillus amylolyticus produces -amylase, pullulanase and -glucosidase. By selection of carbon source in the growth medium, -glucosidase was produced preferentially and with exclusion of the other two activities. The -glucosidase was highly specific for maltose and to a lesser extent maltotriose but was inactive towards a range of other substrates including p-nitrophenyl -D-glucoside and isomaltose. Optima for activity were recorded at pH 7.0 and 40° C and the enzyme was insensitive to ethylenediaminetetraacetic acid.     

N-Carbamoyl-α-Amino Acids Rather than Free α-Amino Acids Formation in the Primitive Hydrosphere: A Novel Proposal for the Emergence of Prebiotic Peptides

Our previous kinetic and thermodynamic studies upon the reactional system HCHO/HCN/ NH₃ in aqueous solutions are completed. In the assumed prebiotic conditions of the primitive earth ([HCHO] and [HCN] near 1 g L^–1, T = 25 °C, pH = 8, [NH₃] very low), this system leads to 99.9% of -hydroxyacetonitrile and 0.1% of -aminoacetonitrile (precursor of the -amino acid). The classical base-catalyzed hydration of nitriles, slow and not selective, can not modify significantly this proportion. On the contrary, we found two specific and efficient reactions of -aminonitriles which shift the initial equilibrium in favor of the -aminonitrile pathway. The first reaction catalyzed by formaldehyde generates -aminoamides, precursors of -aminoacids. The second reaction catalyzed by carbon dioxide affords hydantoins, precursors of N-carbamoyl--aminoacids. In the primitive hydrosphere, where the concentration in carbon dioxide was estimated to be higher than that of formaldehyde, the formation of hydantoins was consequently more efficient. The rates of hydrolysis of the -aminoacetamide and of the hydantoin at pH 8 being very similar, the synthesis of the N-carbamoyl--amino acid seems then to be the fatal issue of the HCHO/HCN/NH₃ system that nature used to perform its evolution. These N-protected -amino acids offer new perspectives in prebiotic chemistry, in particular for the emergence of peptides on the prebiotic earth.     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

Complete protection against melanoma in absence of autoimmune depigmentation after rejection of melanoma cells expressing α(1,3)galactosyl epitopes

The major barrier for xenotransplantation in humans is the presence of (1–3) Galactosyl epitopes (Gal) in xenogeneic tissue and the vast quantities of natural antibodies (Ab) produced by humans against this epitope. The binding of anti-Gal Ab to cells expressing Gal triggers a complement-mediated hyperacute rejection of target cells. The hyperacute rejection of whole cancer cells, modified to express Gal epitopes, could be exploited as a new cancer vaccine to treat human cancers. We tested this hypothesis in Galactosyltransferase knockout (GT KO) mice which, like humans, do not express Gal on their cell surfaces and can produce anti-Gal Ab. Forty-five percent of mice with preexisting anti-Gal Ab rejected Gal positive melanoma cells (B16Gal). These mice remained tumor-free for more than 90 days. The majority of control mice injected with B16Null, Gal negative cells succumbed to melanoma. The rejection of B16Gal induced strong long-lasting antitumor immunity against B16Null measured by the expansion of cytotoxic T lymphocytes. In addition, mice rejecting B16Gal were protected against melanoma since they survived a second rechallenge with B16Null. Protected mice developed antitumor immunity in the absence of autoimmune depigmentation (vitiligo). These results show that rejection of Gal positive melanoma cells can efficiently boost the immune response to other tumor associated antigens present in Gal negative melanoma cells. This study supports the concept of a novel anticancer vaccine to treat human malignancies.     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

Confirmation of assignment of the human α1-crystallin gene (CRYA1) to chromosome 21 with regional localization to q22.3

Summary The crystallins are highly conserved structural proteins universally found in the eye lens of all vertebrate species. In mammals, three immunologically distinct classes are present, -, -, and -crystallins, and each class represents a multigene family. The -crystallin gene family consists of 1-crystallin (CRYA1) and 2-crystallin (CRYA2) genes (previously designated A-and B-crystallin, respectively), which show extensive sequence homology. We constructed a synthetic oligonucleotide probe of 25 bases corresponding to a specific region of the human 1-crystallin gene sequence. This 25-mer probe bears little sequence homology to human 2-crystallin gene and does not cross-hybridize to 2-crystallin sequences in Southern blot analysis. Using this unique synthetic probe, we have demonstrated the identity of the 1-crystallin gene in human genomic DNA. In addition, we have also confirmed its chromosomal location on human chromosome 21. Finally, we have regionally localized the gene to q22.3 by using both Southern blot analysis of a panel of cell hybrids containing different parts of human chromosome 21, and in situ hybridization to metaphase chromosomes. The use of synthetic oligonucleotide probes specific for individual genes should be useful in identifying and mapping members of multigene families.     

Non-radioactive assay and HPLC analysis of cephalosporin C 7α-methoxylation by cell-free extracts of Streptomyces clavuligerus

Summary Escherichia coli W3110 was found to be 50 times more sensitive to cephamycin C than to cephalosporin C, and also markedly more sensitive to 7-methoxycephalosporin C than to 7-hydroxycephalosporin C. Accordingly, this organism could be used to establish a bioassay of cephalosporin C 7-methoxylation by a Streptomyces clavuligerus cell-free extract. The bioassay results were complemented by HPLC analysis. Modification of the mobile phase from 100 mm NaH₂PO₄, pH 4.2, to 200 mm, pH 4.0, improved HPLC resolution such that an unidentified peak could be separated from the 7-hydroxycephalosporin C peak, and the formation of this intermediate as well as the product, 7-methoxycephalosporin C, could be directly measured in the cell-free reaction system. Offprint requests to: A. L. Demain     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Expression of receptor protein tyrosine phosphatase agr; mRNA in human prostate cancer cell lines

Receptor protein tyrosine phosphatase (RPTP) is transmembrane protein phosphatases, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTP mRNA in several normal human tissues. We further investigated the regulation of expression of RPTP mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5-dihydrotestosterone (DHT) down-regulates RPTP mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTP mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTP as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTP mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTP is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

The influence of some natural cell-wall derived precursors on organogenesis and differentiation of wild strawberry (Fragaria vesca L.) callus cultures

Several kinds of precursors obtained by natural polymer hydrolysis of leaves, cell walls, cellulose and hemicelluloses have been tested on in vitro cultures. Interesting preliminary results were obtained with strawberry callus culture whereby all the tested hydrolysates were able to induce differentiation and organogenesis without addition of any plant growth regulators to the culture medium. This suggests the existence of one or more compounds, enzymatically released from the cell wall, which play a role on plant differentiation and organogenesis.Abbreviations AH agar hydrolysate - BA 6-benzyladenine - CH cotton hydrolysate - 2.4-d 2.4-dichlorophenoxyacetic acid - LH leaf hydrolysate - MS Murashige & Skoog medium - NAA -naphthalene-acetic acid - WH cell wall hydrolysate