Amino acid and vitamin requirements in mammalian cultured cells

Summary The development of the tissue culture technique has enabled us to cultivate mammalian cells in a way which is similar to that in use with bacterial cells. As such, the nutritional requirements of mammalian cells in culture have been studied with simplicity and exactness. According to Eagle's extensive works it is accepted that cultured cells generally require 13 amino acids, 8 or 9 vitamins, glucose and 6 inorganic salts. However, although some cultured cells have a capacity for the biosynthesis of Eagle's essential nutrients and others require non-essential nutrients.In this review we will discuss the amino acid and vitamin requirements of cultured cells, and a cell line (R-Y121B · cho) which propagates continuously in a chemically defined medium containing 11 amino acids, 7 vitamins, glucose and 6 ionic salts. Arginine, glutamine, tyrosine and choline are synthesized in the R-Y121B · cho cells.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Plasmodium falciparum: continuous cultivation in a semiautomated apparatus.

A semi-automated apparatus for the continuous cultivation of the malarial parasite, Plasmodium falciparum, was developed. It changes the culture medium and redistributes ths infected erythrocytes at preselected intervals. Parasitemias between 2 and 16% can be maintained by adding fresh erythrocytes every 2 or 3 days. This apparatus produces approximately 10 ml of packed erythrocytes per week with parasitemias between 12 and 16%     

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.     

Reduction of myocardial infarct size by doxycycline: A role for plasmin inhibition

Myocardial ischemia-reperfusion (I/R) is associated with the activation of matrix metalloproteinases (MMPs) and serine proteases. We hypothesized that activation of MMPs and the serine protease plasmin contribute to early cardiac myocyte death following I/R and that broad-spectrum protease inhibition with doxycycline (DOX) preserves myocyte viability. Rats treated daily with or without DOX beginning 48 h prior to experimentation were subjected to 30 min of coronary occlusion and 2 days of reperfusion. DOX pre-treatment reduced infarct size by 37%. DOX attenuated increases in MMP-9 and plasmin levels as determined by gelatin zymography and immunoblot, respectively. Neutrophil extravasation was unaltered by DOX as assessed by myeloperoxidase (MPO) activity. To examine the contribution of MMP-9 and plasmin to myocyte injury, cultures of neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 83 kDa MMP-9 or plasminogen in the presence or absence of DOX. MMP-9 treatment did not affect myocyte viability. Plasminogen treatment led to increased plasmin activity, resulting in loss of ₁-integrin, NRVM detachment and apoptosis. DOX co-treatment inhibited plasmin activity and preserved NRVM attachment, whereas co-treatment with the broad-spectrum MMP inhibitor GM6001 had no effect. These results indicate that plasmin causes disruption of myocyte attachment and viability independently of MMP activation in vitro and that inhibition of plasmin by DOX may reduce I/R-induced myocyte death in vivo through the inhibition of plasmin. (Mol Cell Biochem 270: 1–11, 2005)     

The effect of VDR gene polymorphisms and vitamin D level on blood pressure,risk of preeclampsia,gestational age,and body mass index

We investigated the influence of vitamin D receptor (VDR) polymorphisms and vitamin D level on the blood pressure and the risk of preeclampsia. In a case-control study, 200 pregnant women, including 100 individuals with preeclampsia along with 100 healthy pregnant women, were studied for VDR FokI, TaqI, and BmsI polymorphisms and serum 25 (OH)-D level using polymerase chain reaction-restriction fragment length polymorphism method and commercial kit, respectively. The mean level of 25 (OH)-D in preeclamptic patients was significantly lower (16.6 ± 4.2 ng/mL, P < 0.001) compared with controls (19.6 ± 3.8 ng/mL). Among all women, a significantly higher systolic blood pressure and before-pregnancy body mass index and also lower gestational age were observed in the presence of 25 (OH)-D level < 20 ng/mL compared with the 20 to 30 ng/mL. A significantly higher frequency of VDR FokI C allele in preeclamptic patients (83%) than controls (74%) was associated with a 1.72-fold increased risk of preeclampsia. In all the studied individuals, the systolic and diastolic blood pressures were significantly higher in the presence of the FokI CC genotype compared with the TC and TT+TC genotypes. Neither VDR Taq1 nor VDR BmsI was associated with the risk of preeclampsia. The haplotype FokI C, TaqI C and BmsI A (CCA) compared with haplotype CTG increased the risk of preeclampsia by 1.4-fold (P = 0.33). Our study suggests an association between VDR FokI polymorphism and an insufficient serum level of 25 (OH)-D with the risk of preeclampsia and also the influence of insufficient 25 (OH)-D level and VDR FokI polymorphism on maternal factors, including blood pressure.