Hypoxia reduces expression and function of system A amino acid transporters in cultured term human trophoblasts

We tested the hypothesis that hypoxia diminishes the expression and transport of neutral amino acids by system A in full-term human trophoblasts. Cytotrophoblasts from normal human placentas were cultured in standard conditions of 20% O(2) or in 1% and 3% O(2) for 24 h before assay. Neutral amino acid transport for systems A, ASC, and L was assayed at 24 and 72 h by the cluster-tray technique. Hypoxia during the initial 24 h of culture reduced system A transport by 82% in 1% O(2) and by 37% in 3% O(2) (P < 0.01) compared with standard conditions. Hypoxia during the latter 24 h of the 72 h in culture reduced system A transport by 55% in 1% O(2) and by 20% in 3% O(2) (P < 0.05) compared with standard conditions at 72 h. Hypoxia (1% O(2)) also reduced total amino acid transport by 40% in the more differentiated syncytiotrophoblasts present at 72 h. Northern analysis of trophoblasts in standard conditions showed that subtypes of human amino acid transporter A (hATA1 and hATA2) were each expressed in cytotrophoblasts and syncytiotrophoblasts. Hypoxia decreased expression of hATA1 and hATA2 in both trophoblast phenotypes. We conclude that hypoxia downregulates system A transporter expression and activity in cultured human trophoblasts.     

Activation of metastatic potential in african green monkey kidney cell lines by prolongedin vitro culture

Summary Studies on the tumorigenicity of Vero kidney cells ofCercopithecus aethiops monkey origin were extended to various passage levels of BSC-1 aneuploid cells and to low passage CV-1 diploid cells (derived also fromC. aethiops monkey kidney). It was found that BSC-1 cells—like Vero cells—showed increased tumorigenicity with increasing passage level in antitymocyte globulins (ATG) treated newborn rats and in nude mice. Cells passaged over 250 times in cultures formed invasive adenocarcinomas in newborn rats. Their malignant tumor growth was further demonstrated around the 500 passage level when tumor metastases were detected in the lungs of four of the 14 inoculated rats. Vero cells induced such lung metastases in rats already at passage 227. CV-1 diploid cells at low passage level produced small nodules of epithelioid cells in newborn rats at 6th day after inoculation that had disappeared by the 21st day, and caused no local invasion nor lung metastasis. In vitro tumorigenicity tests on BSC-1 and CV-1 cells, using chick embryo skin, human muscle and colony formation in agarose, confirmed the animal test results. The results of this study indicate that BSC-1 and Vero cell lines at low and high passage levels may prove to be useful tools to study the molecular basis of malignancy. Editor's Statement In this paper Contreras et al. document the increased malignancy of commonly-used monkey cell lines upon long-term culture. These observations have implications for the use of these cell lines in studies of cancer cell biology, as well as the use of these lines for the production of biologicals. David W. Barnes     

Genetic characterization of Sabin types 1 and 3 poliovaccine virus following serial passage in the human intestinal tract.

Poliovirus isolates types 1 and 3 were obtained from five and seven successive passages respectively, in infants who had been fed monovalent OPV in two separate clinical trials conducted in 1960. The purpose of these trials was to answer the question how much the vaccine virus would revert to its original neurovirulent phenotype following multiplication in the intestinal tract. Human passages were performed either by contact exposure or by feeding the excreted virus while the infants were maintained in isolation. Several virus isolates were obtained at each passage level. Infants participating in both studies showed no symptoms of disease. Antigenic studies (McBride, van Wezel) and protein analysis (PAGE) of the isolates, reported earlier from this laboratory, had shown that the isolates remained vaccine-like, although isolates from the later passages revealed some differences. Monkey neurovirulence test results showed that for both types 1 and 3 viruses the loss of attenuation of the vaccine strain upon passage was gradual, although the loss was faster for type 3. Examination of the oligonucleotide maps demonstrated that the oligonucleotide configuration of the isolates remained the same as for the vaccine strain but there was an increase of individual spot differences with increasing passage. The nucleotide sequence analysis of selected regions of the virus genomes revealed that there was no change from a G to A in nucleotide 480 of type 1 isolates; however, nucleotide 476 changed from a U to an A in type 1 passages 3, 4 and 5. Conversely, for type 3 the change of nucleotide 472 from a U to a C changed at the early first passage (4 days following administration of OPV), and remained a C in the six following passages; type 3 nucleotide 2034 did not change in the first passage from a U to a C, but it became a C in all further passages tested. The nucleotide changes mentioned for both virus types remained stable in successive passages. However, there was another nucleotide change for type 3 from a U to a C at position 1973 only for passages 5 and 6 which reverted to a U for passages 7L and 7LL. Study of selected human passage virus strains could further contribute to the identification of the critical nucleotides that are responsible for the attenuation of these two polio types of vaccine viruses.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Blood groups and protein polymorphisms in five goat breeds (Capra hircus)

Data on allele frequencies at six red cell blood group systems and three blood protein polymorphic loci in five goat breeds are reported. Two blood proteins, albumin and carbonic anhy-drase, were not found to be polymorphic. The B blood group system of goats, like its homologue in cattle and sheep, is highly complex. At least 44 B phenogroups (haplotypes) have been distinguished in this study. Based on the variation in allele frequencies between breeds, genetic distances were calculated. The distances estimated by four different methods were in close agreement with data from the history and geographic origins of the breeds examined.