Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.     

Maternal influence on activity rhythms and reproductive development in Djungarian hamster pups

Photoperiodism and entrainment of the circadian rhythm of locomotor activity were investigated in juvenile Djungarian hamsters. Animals were housed in simulated burrows. Activity was measured as the animal's emergence from a dark nest chamber into an outer box exposed to the room illumination. This burrow emergence activity exhibited marked circadian rhythmicity. Interactions between mother hamsters and their offspring were examined in the simulated burrow system. Male reproductive responses were determined by measuring testicular weights at the time of weaning. It was shown that photoperiodic information received between Days 1 and 15 of life failed to alter the rate of testicular development, but that after Day 15 testicular growth was photoperiod-dependent. The mother, when entrained to a long photoperiod, did not influence the photoperiodic responses of her pups when they were confined to a dark nest box. In contrast, the mother did influence the circadian entrainment patterns of her pups. Pups exhibited a well-developed circadian activity rhythm at weaning with a phase angle roughly similar to that of the mother's activity rhythm. When the maternal rhythms were discrepant with photoperiod information received by the pups directly from the environment, the pups' activity rhythms were synchronized with the light/dark cycle rather than with the rhythm of their mother. Thus, it appears that although pups may first become entrained by maternal cues, they rapidly adjust to the environmental light cycle after leaving the nest.     

Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms

Twenty isolates of the dimorphic, pathogenic fungus Histoplasma capsulatum were divided into three classes based on comparisons of restriction enzyme digests of their mitochondrial DNA and rDNA. The majority of isolates, including most North American strains and the African H. capsulatum var. duboisii variants, belong to class 2. Isolates from Central America and South America make up class 3. The attenuated Downs strain is the only member of class 1.     

Aspartic acid administered neonatally affects ventilation of male and female rats differently

In this study ventilation was evaluated in 12-mo-old male and female rats who had received large doses of aspartic acid neonatally. Rats of both sexes treated with aspartic acid were obese, stunted, and exhibited hypogonadism. Although metabolic rates of the aspartic acid-treated rats were not different compared with sex-matched controls, ventilatory patterns were different. Aspartic acid-treated females breathed with a smaller tidal volume (VT), higher frequency (f), and similar minute ventilation (VE) compared with control females. This pattern is commonly observed in many patients who are obese. The aspartic acid-treated females responded to hypercapnic and hypoxic challenges by increasing f more than VT. Tissue pocket gases (PCO2 and PO2) of aspartic acid-treated females were normal. In contrast, aspartic acid-treated males hypoventilated compared with control males. Tissue pocket gas values suggested that aspartic acid-treated males were hypoxemic and hypercapnic. Moreover, the response of aspartic acid-treated males to hypercapnia was parallel to but was less than that of control male rats. The ventilatory response of aspartic acid-treated male rats to hypoxia was blunted. This study has shown that neonatal administration of aspartic acid causes a decreased ventilation and blunted response to hypoxia in adult male but not female rats.     

Genomic DNA sequence for human C-reactive protein

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.     

Twenty-seven protein polymorphisms by two-dimensional electrophoresis of serum, erythrocytes, and fibroblasts in two pedigrees

Twenty-seven independent polymorphic loci were detected by two-dimensional electrophoresis (2DE) of serum, erythrocytes, and fibroblasts in two large families and analyzed for linkage to classical genetic markers. We detected seven serum, four erythrocyte, and 17 fibroblast protein loci that exhibited charge variation in these two families and in a sample of unrelated individuals. The genetic basis of protein variants was confirmed by quantitative gene-dosage dependence and by conformance to Mendelian transmission in the two families, except for four rare variants for which transmission analysis was not possible. Linkage analysis demonstrated that each of the variants represent products of independent loci, with the exception of erythrocyte locus (RBC4), which we also detected in fibroblasts (NC27). Two allozyme polymorphisms, glyoxalase-1 (GLO1) and phosphoglucomutase-3 (PGM3) were specifically identified here based on genotypic concordance and molecular mass. Unknown fibroblast protein (NC22) may be linked to apolipoprotein E (lod score = 2.8 at theta m = theta f = 0), while a serum protein locus (SER1) may be linked to alpha-haptoglobin (lod score = 2.54 at theta m = .20, theta f = .01). Six of seven polymorphic serum loci were previously located on two-dimensional gels: alpha-1 antitrypsin (PI), Gc-globulin (GC), alpha-2 HS glycoprotein (HSGA), alpha-haptoglobin (HP), and two apolipoproteins (APOE and APOA4). Six of 17 polymorphisms detected in fibroblasts were positionally identical to polymorphic loci seen in lymphocytes. These studies indicate a minimum level of average protein charge heterozygosity of approximately 2.2% for the most predominant human cellular proteins and of 5.6% for the most predominant proteins of serum.     

Dexamethasone-induced phospholipase A2-inhibitory proteins (PLIP) influenced by the H-2 histocompatibility region

Recent work has indicated that the H-2 histocompatibility complex on chromosome 17 influences the degree of glucocorticoid-induced teratogenicity and anti-inflammatory response. Since both of these hormonal actions appear to be mediated by the induction of phospholipase A2-inhibitory proteins (PLIP), the influence of the H-2 complex on the induction of PLIP by glucocorticoids in thymocytes and embryonic palates has been investigated. Analysis of dexamethasone-induced PLIP by Sephadex G-100 revealed four peaks of mol wt 55,000, 40,000, 28,000 and 15,000 in mouse thymocytes and from one to three of these PLIPs in mouse embryonic palates. The 55,000 mol wt PLIP comprised 50-60% of the total activity. The total amount of dexamethasone-induced PLIP is significantly higher in B10.A (H-2a) thymocytes than that in thymocytes of their congenic resistant partners, B10 (H-2b). The induced level of PLIP in the embryonic palates treated with dexamethasone is also significantly higher in the H-2a congenic strains with either the A or B background (AWy or B10.A) than that in their resistant partners (A.BY or B10). Thus, both susceptibility to glucocorticoid-induced cleft palate and the production of PLIP by this hormone are influenced by the H-2 complex.