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Many important changes occur throughout reproductive development in beef heifers, including during the prenatal, early postnatal, and peripubertal periods. Wave-like patterns of follicular development have been observed in heifer calves as early as 2 wk of age. Some dramatic changes occur from about 2 to 5 mo of age, most notably the transient increase in LH secretion. Most components of the hypothalamic-pituitary-ovarian axis are fully competent by approximately 5 to 6 mo of age. Peripubertal changes include increases in LH secretion, estradiol production, follicular development, and reproductive tract size. Eventually, the process reaches the point that the initial ovulation is achieved. Heifers that reach puberty and experience multiple estrous cycles before the onset of their initial breeding season have a greater probability for early conception and optimal lifetime productivity. Attainment of puberty typically occurs at around 12 to 14 mo of age in beef heifers but varies greatly. Genetic differences and environmental factors contribute to this variation. In typical U.S. cow-calf operations, calves are generally weaned at approximately 200 d of age. The impact of postweaning management on age at puberty in heifers has been demonstrated, and there is considerable flexibility in the timing of gain from weaning to breeding. However, even when heifers are grown to the desired BW before the start of breeding, there remains a pronounced variation in the timing of puberty, which impacts pregnancy rates. Less attention has been focused on the impact of preweaning management on age at puberty. Heifer calves with increased growth rates from birth to weaning have reached puberty at earlier ages. Precocious puberty has also been induced in a majority of heifers with early weaning and feeding a high-concentrate diet. Nutritional control during early maturation in heifers exerts a substantial influence on the timing of puberty. Understanding the mechanisms involved in reproductive development increases our ability to effectively manage replacement beef heifers for optimal reproductive performance.

We tested the hypothesis that luteal function and fertility would be reduced in cattle induced to ovulate prematurely compared with those ovulating spontaneously. Estrus was synchronized in 56 beef cows (24 that were nonlactating and 32 that were nursing calves). At 6.4 ± 0.1 d after estrus, all follicles ≥5 mm were aspirated (day of aspiration = d 0) with a 17-gauge needle using the ultrasound-guided transvaginal approach. On d 1.5 and 2, cows were administered 2 luteolytic doses of PGF2α. Ovarian structures were monitored by transrectal ultrasonography from d −2 to 12, or ovulation. Emergence of a new follicular wave occurred on d 1.7 ± 0.1. When the largest follicle of the newly emerged wave was 10 mm in diameter (d 4.8 ± 0.1), cows were assigned on an alternating basis to receive 100 μg of GnRH (GnRH-10; n = 29) to induce ovulation or, upon detection of spontaneous estrus, to the spontaneous (SPON) treatment (n = 24). Cows were bred by AI at 12 h after GnRH (GnRH-10) or 12 h after the onset of estrus (SPON) as detected using an electronic surveillance system. Blood samples were collected every other day beginning 2 d after ovulation until pregnancy diagnosis 30 d after AI. Ovulation and AI occurred in 29/29 cows in the GnRH-10 and in 24/24 cows in the SPON treatment. Ovulation occurred later (P< 0.05) in the SPON (d 7.7 ± 0.1) than GnRH-10 (d 6.8 ± 0.1) treatment. Double ovulations were detected in 47% of cows, resulting in 1.5 ± 0.1 ovulations per cow. Diameters of the ovulatory and the second ovulatory (in cows with 2 ovulations) follicles were greater (P< 0.05) in the SPON (12.0 ± 0.3 mm and 10.5 ± 0.4 mm, respectively) than in the GnRH-10 (10.7 ± 0.1 mm and 9.2 ± 0.3 mm) treatment. Cross-sectional areas of luteal tissue and plasma concentrations of progesterone during the midluteal phase were greater (P< 0.05) in the SPON (3.62 ± 0.2 cm2and 6.4 ± 0.3 ng/mL) than in the GnRH-10 (3.0 ± 0.2 cm2and 5.4 ± 0.2 ng/mL) treatment. The conception rate to AI in the SPON (100%) treatment was greater (P< 0.05) than in the GnRH-10 (76%) treatment. The animal model used in this study resulted in unusually high conception rates and double ovulations. In conclusion, premature induction of the LH surge reduced the diameter of ovulatory follicle(s), the luteal function, and the conception rate to AI.

Precocious puberty (<300 d of age) can be successfully induced in a majority of heifers with early weaning and continuous feeding of a high-concentrate diet. The objective of this experiment was to determine the relative effects of timing of feeding a high-concentrate diet on age at puberty in early-weaned heifers. Sixty crossbred Angus and Simmental heifer calves were weaned at 112 ± 2 d of age and 155 ± 3 kg of BW and were fed a receiving diet for 2 wk. Heifers were blocked by age and BW, and assigned randomly to receive a high-concentrate (60% corn; H) or control (30% corn; C) diet during phase 1 (mean age 126 to 196 d) and H or C during phase 2 (mean age 196 to 402 d), resulting in 4 treatments (HH, n = 15; HC, n = 15; CH, n = 15; and CC, n = 15). Blood samples were collected weekly beginning at a mean age of 175 d and assayed for progesterone concentration to determine age at puberty. After 56 d on the experimental diets, BW of heifers fed the H diet during phase 1 were greater (P< 0.05) than those of heifers fed the C diet (mean age of 182 d; treatment × mean age, P< 0.01). After 70 d on the new diets (mean age of 266 d), heifers fed the H diet during phase 2 reached heavier BW (P< 0.05) than heifers fed the C diet, when compared within phase 1 diet groups (HH > HC; CH > CC). Body weights in HC and CH treatments differed from a mean age of 169 through 238 d, after which BW did not differ between these treatments. The ADG over the entire experimental period was greatest for the HH treatment (1.2 ± 0.04 kg/d; P< 0.05), followed by the HC and CH treatments (1.0 ± 0.03 and 1.0 ± 0.02 kg/d, respectively), which were not different, and the CC treatment gained the least (0.7 ± 0.04 kg/d; P< 0.05). Precocious puberty occurred in 67, 47, 47, and 20% of heifers in the HH, HC, CH, and CC treatments, respectively (HH > CC; P< 0.05). Mean age at puberty for the HH and HC treatments (271 ± 17 and 283 ± 17 d of age, respectively) was earlier (P< 0.05) than for the CC treatment (331 ± 11 d of age). Age at puberty in the CH treatment (304 ± 13 d of age) was intermediate to and not different from the other treatments. Heifers fed the H diet during phase 1 attained puberty earlier (P< 0.05) than heifers fed the C diet during phase 1. In conclusion, increasing dietary energy intake in early-weaned heifers, through feeding a high-concentrate diet from 126 to 196 d of age, decreased age at puberty regardless of the diet fed after 196 d of age.

Precocious puberty can be induced in a majority of heifers weaned early and fed a high-concentrate diet. The objective of this experiment was to determine whether induction of precocious puberty is associated with an acceleration of ovarian maturation in heifers. Crossbred Angus and Simmental heifer calves were weaned at 104 ± 2 (n = 18; early weaned) or 208 ± 3 (n = 10; normal-weaned, NW) d of age. The early weaned heifers were fed a high-concentrate (60% corn; EWH, n = 9) or control diet (30% corn; EWC, n = 9). The NW heifers were also fed the control diet after weaning. Daily transrectal ultrasonography was performed to characterize a complete follicular wave beginning at a mean age of 126, 161, 196, 224, and 252 (EWH and EWC), or 224 and 252 (NW) d. Blood samples were collected daily during periods of ultrasonography to determine estradiol concentrations and weekly beginning at mean ages of 153 (EWH and EWC) or 216 (NW) d to be analyzed for progesterone concentrations. Heifers in the EWH treatment were heavier (P< 0.01) than EWC heifers from a mean age of 175 d through the end of the study (treatment × age; P< 0.05). Body weights did not differ between EWC and NW. At mean ages of 196 and 224 d, the maximum diameter of the dominant follicle (MaxDF) was greater (P< 0.05) in EWH than EWC heifers. At a mean age of 224 d, MaxDF was greater (P< 0.05) in EWC than NW heifers but was not different by a mean age of 252 d. All EWH, 5 of 9 EWC, and 5 of 10 NW heifers attained puberty at less than 300 d of age (precocious puberty). Age at puberty was less (P< 0.05) in EWH (252 ± 9 d) than in EWC and NW (308 ± 26 and 330 ± 25 d, respectively) treatments. Across all heifers, MaxDF and duration of follicular waves increased with age (P< 0.05), mean number of follicles during follicular waves decreased with age (P< 0.05), and peak concentrations of estradiol during follicular waves increased until a mean age of 224 d. To further characterize aspects of precocious puberty, heifers were compared across treatments between those that experienced precocious puberty and those that did not. In heifers that experienced precocious puberty, BW at puberty was less (P< 0.01) and MaxDF, follicular wave duration, and peak estradiol concentrations were greater (P< 0.05) compared with heifers that did not experience precocious puberty. Ovarian maturation was accelerated in heifers that were weaned early and fed a high-concentrate diet and was associated with precocious onset of puberty.

In beef heifers weaned between 3 and 4 mo of age and fed a high-concentrate diet, approximately 50% reach puberty before 300 d of age (precocious puberty). The objectives of this experiment were 1) to determine whether precocious puberty could be induced experimentally by weaning heifers early and feeding a high-concentrate diet, and 2) to determine the dynamics of secretion of LH associated with precocious puberty. Crossbred Angus and Simmental heifer calves were weaned at 73 ± 3 d of age and 115 ± 3 kg of BW and fed a high-concentrate (60% corn; HI, n = 9) or control diet (30% corn; CONT, n = 9). Heifers were fed individually, and target BW gains were 1.50 and 0.75 kg/d for the HI and CONT treatments, respectively. Heifers were weighed every 2 wk. Blood samples were collected weekly and assayed for progesterone concentration to determine age at puberty. Serial blood samples were collected at 20-min intervals for 24 h at mean ages of 102, 130, 158, 172, 190, 203, 217, 231, and 259 d and assayed for LH concentration to evaluate the dynamics of secretion of LH. Heifers fed the HI diet exhibited greater BW gain (P< 0.01) than CONT heifers (1.27 ± 0.05 vs. 0.85 ± 0.05 kg/d, respectively). As a result, BW in the HI treatment was greater (P< 0.01) than in the CONT treatment by 188 d of age and remained different through the end of the experiment. Precocious puberty occurred in 8 of 9 heifers fed the HI diet and 0 of 9 heifers fed the CONT diet. Age at puberty was reduced in the HI (P< 0.01) compared with the CONT heifers (262 ± 10 vs. 368 ± 10 d of age, respectively). Body weight at puberty was also reduced in the HI (P< 0.05) compared with the CONT treatment (327 ± 17 vs. 403 ± 23 kg, respectively). Heifers attaining puberty during the experiment continued with subsequent luteal phases as evidenced by cyclic patterns of progesterone concentrations. Frequency of pulses of LH (pulses/24 h) increased with age (P< 0.01) for both treatments. Heifers in the HI treatment exhibited a greater number of pulses of LH (P< 0.01) than those in the CONT treatment by 190 d of age and in all subsequent collection periods (treatment × age, P< 0.05). Mean LH concentrations also increased with age (P< 0.01) for both treatments but did not differ between treatments. In conclusion, precocious puberty induced by early weaning and feeding of a high-concentrate diet is preceded by increasing frequency of pulses of LH.

Precocious puberty (<300 d of age) can be induced in beef heifers by early weaning and feeding a high-concentrate diet. The objective of this experiment was to determine whether precocious puberty occurs as a result of a hastened reduction of estradiol negative feedback on secretion of LH. Thirty crossbred Angus and Simmental heifers were weaned at 83 ± 2 d of age and 114 ± 3 kg of BW, blocked by BW, and randomly assigned to receive a high-concentrate (60% corn; H) or control (30% corn; C) diet and to receive ovariectomy (OVX), OVX plus an estradiol implant (OVXE), or to remain intact (INT). Residual ovarian tissue after OVX necessitated withdrawal of 6 heifers during the course of the experiment, resulting in the following treatment groups: OVX-C, n = 3; OVX-H, n = 5; OVXE-C, n = 4; OVXE-H, n = 2; INT-C, n = 5; INT-H, n = 5. To determine concentrations of progesterone and estradiol, blood samples were collected weekly beginning at a mean age of 160 d. To characterize LH concentrations, serial blood samples were collected at 12-min intervals for 12 h at mean ages of 119, 149, 188, 217, 246, 281, 323, 365, 407, and 449 d. By a mean age of 202 d, heifers fed the H diet were heavier (P< 0.05) than those fed the C diet. Heifers in the INT-H treatment attained puberty earlier (P< 0.05) than in the INT-C treatment (275 ± 30 vs. 385 ± 14 d of age, respectively). Overall mean concentrations of estradiol did not differ between OVXE-H and OVXE-C, between INT-H and INT-C, or between OVXE and INT treatments. The OVX treatments exhibited greater LH pulse frequency than the OVXE and INT treatments by the first serial blood collection (treatment × age, P< 0.05). The frequency of LH pulses was greater (P< 0.05) in the INT-H than the INT-C treatment by a mean age of 246 d and was greater (P< 0.05) in the OVXE-H than the OVXE-C treatment by a mean age of 281 d. In the OVXE-H treatment, LH secretion increased and subsequently “escaped” from estradiol negative feedback (detection of ≥1 LH pulse/h) earlier (P< 0.05) than in the OVXE-C treatment (307 ± 30 and 420 ± 21 d of age, respectively). It is concluded that advancing the reduction of estradiol negative feedback on secretion of LH is the mechanism by which early weaning and feeding a high-concentrate diet results in precocious puberty in heifers.

Previous analyses of tso1 mutants revealed a loss of control of directional cellular expansion and coordination of growth of adjacent cells, and defects in karyokinesis and cytokinesis. We isolated TSO1 using a map-based approach, and show that it is a member of a family of at least three genes in Arabidopsis. Consistent with the mutant phenotype, TSO1 transcript was most abundant in flowers, where it accumulated to the highest levels in developing ovules and microspores. The putative TSO1 protein has two cysteine-rich regions that are similar to the CXC domains of a variety of proteins from plants and animals, including a class of kinesins involved in chromosome segregation, and enhancer of zeste-type proteins. Visualization of TSO1-fusion proteins indicated that TSO1 is a nuclear protein. The tso1 mutant phenotypes and the novelty of the TSO1 sequence suggest the existence of previously unknown participants in regulation of directional processes in eukaryotic cells.

The Arabidopsis INNER NO OUTER (INO) gene is essential for formation and asymmetric growth of the ovule outer integument. INO encodes a member of the newly described YABBY family of putative transcription factors that contain apparent Cys(2)-Cys(2) zinc-finger domains and regions of similarity to the high mobility group (HMG) transcription factors. In wild-type plants, INO is expressed specifically on one side of the central region of each ovule primordium in the cells that give rise to the outer integument. Alterations in the INO expression pattern in mutant backgrounds implicate INO as a positive regulator of its own expression, and ANT, HLL, BEL1, and SUP as direct or indirect negative regulators that help to establish the spatial pattern of INO expression. We hypothesize that INO is necessary for polarity determination in the central part of the ovule. Maintenance of polarity in other parts of ino ovules indicates the existence of additional regulators and provides further evidence that the ovule is a compound structure.

The synthesis of Tl2Ba2CaCu2O8(2212) films obtained byex situannealing of amorphous films, deposited at room temperature with a multitarget sputtering system, is described. The growth phenomenon of 2212 films has been extensively studied by annealing precursor thin films of variable thallium content under various O2partial pressures. The best single-phase 2212 films are obtained from stoichiometric amorphous precursor films. Such 2212 films, annealed under low oxygen pressure, exhibit a critical temperature for superconductivityTc(Resistivity = 0) at 110K with a critical current densityJc(77 K) > 106A/cm2. After annealing, the composition of these films, deduced from the backscattering spectra of4He+beam measurements, indicates a formula close to Tl:Ba:Ca:Cu = 2:2:1:2.

cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coli. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.

The salt-sensitive phenotype of yeast cells deficient in the phosphoprotein phosphatase, calcineurin, was used to identify genes from the higher plant Arabidopsis thaliana that complement this phenotype. cDNA clones corresponding to two different sequences, designated STO (salt tolerance) and STZ (salt tolerance zinc finger), were found to increased tolerance of calcineurin mutants and of wild-type yeast to both Li+ and Na+ ions. STZ is related to Cys2/His2-type zinc-finger proteins found in higher plants, and STO is similar to the Arabidopsis CONSTANS protein in regions that may also be zinc fingers. Although neither protein has sequence similarity to any protein phosphatase, STO was able to at least partially compensate for all tested additional phenotypic effects of calcineurin deficiency, and STZ compensated for a subset of these effects. Salt tolerance produced by STZ appeared to be partially dependent on ENA1/PMR2, a P-type ATPase required for Li+ and Na+ efflux in yeast, whereas the effect of STO on salt tolerance was independent of ENA1/PMR2. STZ and STO were found to be expressed in Arabidopsis roots and leaves, whereas only STO message was detectable in flowers. An apparent increase in the level of STZ mRNA was observed in response NaCl exposure in Arabidopsis seedlings, but the level of STO mRNA was not altered by this treatment.

We have used the technique of DNA-mediated gene transfer to introduce dihydrofolate reductase genes into dihydrofolate reductase-deficient Chinese hamster ovary cells. The transferred sequences include: dihydrofolate reductase minigenes, dihydrofolate reductase cDNA clones, and genomic DNA from mouse cells with highly amplified dihydrofolate reductase genes. The hamster cells were capable of utilizing the murine transcription initiation sites, splice junctions, and polyadenylation sites in complete murine dihydrofolate reductase genes. Only a short region of 5'-flanking sequence (160 base pairs (bp] was sufficient for proper initiation of dihydrofolate reductase transcription. In contrast only those clones with all of the dihydrofolate reductase introns and extensive (greater than 6.5 kilobase pairs) 3'-flanking regions utilized the dihydrofolate reductase polyadenylylation sites. Four of nine clones tested regulate the transfected genes normally, synthesizing dihydrofolate reductase preferentially at the onset of S phase. Regulated genes include one which entirely lacks intervening sequences, two with fewer than 420 bp of 5'-flanking sequence, and two in which polyadenylylation occurs predominantly in the adjacent hamster sequences. Two of the five cell lines which do not exhibit normal regulation contain genes which appear to be transcribed primarily from promoters present in the flanking hamster DNA. The other genes which fail to regulate do not appear to differ significantly in the structure of their transcripts or protein products from genes which regulate normally. We conclude that only the coding sequences, a region of less than 340 bp of sequence 5' of the translation initiation codon, and a short region of 3'-flanking sequence are required for regulated production of dihydrofolate reductase. The regulation can be abolished, however, by unknown properties of the site of insertion.

Our understanding of the molecular mechanisms that regulate and integrate the temporal and spatial control of cell proliferation during organ ontogenesis, particularly of floral organs, continues to be primitive. The ovule, the progenitor of the seed, of Arabidopsis thaliana has been used to develop an effective model system for the analysis of plant organogenesis. A typical feature of a generalized ovule is the linear arrangement of at least three distinct elements, the funiculus, chalaza and nucellus, along a proximal-distal axis. This pattern is supposed to be established during the early proliferative phase of ovule development. We provide genetic evidence that the young ovule primordium indeed is a composite structure. Two genes, HUELLENLOS and AINTEGUMENTA have overlapping functions in the ovule and differentially control the formation of the central and proximal elements of the primordium. The results indicate that proximal-distal pattern formation in the Arabidopsis ovule takes place in a sequential fashion, starting from the distal end. Furthermore, we show that HUELLENLOS also regulates the initiation and/or maintenance of integument and embryo sac ontogenesis and interestingly prevents inappropriate cell death in the young ovule.

Tcoptimization of as-synthesized Tl 2212 thin films can be obtained through post-annealing treatments at low temperatures (180 and 220°C). In this paper, the optimization process was controlled by ac magnetic susceptibility measurements for theTc's and by polarized X-ray absorption spectroscopy measurements for the doping hole densities. The latter technique allows a direct insight into the hole density only in the (a,b) plane of the Tl 2212 structure. All the studied thin films are overdoped with respect to the optimum of 0.12 doping holes per copper defined previously.Tcincreases up to 6 K were observed after post-annealing treatments under an argon atmosphere and were correlated with significant reduction of the doping hole density. In contrast to what has been observed on sintered samples, a post-annealing treatment at 220°C resulted in an insufficient doping hole density, below the optimum, probably resulting from the small sample thickness.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase is an enzyme of the shikimate pathway which is located in the chloroplasts in higher plants. This enzyme is the target of the nonselective herbicide glyphosate. We have isolated and sequenced cDNA clones encoding EPSP synthase from petunia and tomato. The deduced amino acid sequences of the two enzyme precursors show 93% identity in the mature protein regions and 58% identity in the transit peptides. The sequences of the plant enzymes show significantly greater similarity to bacterial EPSP synthases than to fungal EPSP synthases. A genomic clone containing an EPSP synthase gene was isolated from a library of petunia DNA and was shown to contain seven intervening sequences. This gene is expressed approximately 25-fold higher in flower petals than in other organs of petunia. Transcription of this gene is initiated at multiple sites in petunia leaves and in a glyphosate-tolerant petunia cell line overproducing EPSP synthase mRNA. In petals, however, transcription of this gene is initiated almost entirely from only one of these sites. In contrast to petunia, the levels of EPSP synthase mRNA in different organs of tomato do not differ significantly.

We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants.

We have isolated clones of an Arabidopsis gene (ROF1, forrotamaseFKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 — a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1.ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance ofROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.

We have constructed a number of abbreviated dihydrofolate reductase (DHFR) genes by using cloned mouse genomic and cDNA sequences. These genes contain 1.0 kilobase of 5' flanking genomic sequence and varying portions of the 3' non-coding region. Two of the genes contain the first two introns of the DHFR gene; the other three lack introns. Transfection of DHFR-deficient Chinese hamster ovary cells with any of these constructed genes results in cells with the DHFR+ phenotype. Treatment of the transfectants with methotrexate, a folate antagonist, leads to the emergence of methotrexate-resistant colonies which have amplified the transfected genes.

In a case of primary atypical pneumonia (blood grounp O), incomplete antibodies of the cold agglutinin type with a very wide thermal spectrum (35 to 37 C.) led to a severe immunopancytopenia in the form of severe hemolytic anemia, agranulocytosis, and thrombocytopenia. It is obvious that hemolysis was not induced by cold agglutinins which were active 1:1024 at 4 C. and only 1:32 at 22 C. The indirect cold antibodies which were probably responsible for the hemolysis which occurred were thermolabile (1:16 at 22 C., 1:8 at 37 C.) and could be demonstrated only in the antiglobulin plasma test. During the acute phase, too, a hemolysin of the Dacie type active for normal erythrocytes, but even more active for trypsinized cells, at 37 C. and at a pH of 6.8 and 7.6 could be demonstrated. The severe hemolysis could be stopped by ACTH treatment but the titer of the complete and incomplete cold antibodies showed no distinct change and the direct and indirect Coombs test remained positive for a long time. However, there was a distinct diminution of the thermal amplitude of the incomplete antibodies. A severe agranulocytosis (140 granulocytes) developed which could be improved each time by cortisone. However, permanent improvement resulted only after the disappearance of the incomplete antibodies one year later. A leukopenic factor could be demonstrated by transfusion of 300 cc. blood to a recipient of the same blood group producing a pronounced fall in the granulocytes of from 4200 to 910. Control transfusions from a normal inidividual to the same recipient showed no change and the same is true for the transfusion experiment performed one year later from the now cured patient to the same recipient. Leukocyte agglutination tests were negative, but it is presumed that agglutinins of the incomplete type may have been present. Opsonins could not be demonstrated. The thrombocytopenia never descended below 50,000. The bone marrow showed a distinct shift to the left, i.e. a predominance of unripe forms. On the basis of our previous experimental work (amidopyrine agranulocytosis) it is believed that the changes in the marrow are brought about by a depletion and exhaustion of the bone marrow due to the enormously increased peripheral destruction of the granulocytes by agglutination rather than to an actual inhibition of the marrow. The relation of this immunoleukopenia in our previous findings to the occurrence of leukocyte agglutination in agranulocytosis of allergic origin is discussed. Agranulocytosis and leukopenia may be produced on an immunologic basis in many other cases and thus some form of agranulocytosis may be related to the mechanism of erythrocyte destruction in acquired hemolytic anemia and to the agglutination of thrombocytes ins essential thrombocytopenia on an immunologic basis.

Highly c-axis and in-plane oriented HgBa2Ca2Cu3Oy(Hg-1223) thick films with a Tc(zero) as high as 130 K have been successfully fabricated on SrTiO3by annealing precursor films prepared by spray pyrolysis in Hg-vapor atmosphere. These 1.5 – 10 μm thick films exhibited a high zero-field transport Jcabove 105A/cm2at 100 K. Magnetization measurements also revealed a high Jceven at 5 T at low temperatures. Application of similar annealing process to HgO/Ba2Ca2Cu3Oymultilayer precursor films prepared by pulsed laser deposition resulted in c-axis oriented Hg-1223 thin films with a Tcof 128 K.