Increased adherence of human granulocytes to herpes simplex virus type 1 infected endothelial cells

Summary We studied the interaction of human polymorphonuclear leukocytes (PMNs) with umbilical vein endothelial cells infected with herpes simplex virus (HSV) type 1. PMNs labeled with⁵¹Cr were added to endothelial monolayers at varying times after infection and their adherence assessed 1 h later. Granulocyte adherence (GA) to uninfected cells averaged 26.5±1.9%. Increased adherence began 6 h postinfection and rose to a maximum at 20 to 24 h. HSV-1 glycoproteins seemed to mediate the increase in GA: tunicamycin treatment of infected monolayers for 18 h abolished the increased GA as did incubation of infected cells with F(ab')₂ fragments prepared from human antiserum containing HSV-1 antibody. Supported by grants R01-AA-06029 and T32-AA07233 from the National Institute of Alcohol Abuse and Alcoholism, and R01-HL-28220 from the National Heart, Lung, and Blood Institute.     

Factors influencing microbiological assay of cell-culture mycoplasmas

Summary A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%).M. orale andA. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aerobic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains ofM. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas. These studies were supported in part by Contracts N01-AG-4-2865 and N01-AG-8-2117 from the National Institute on Aging and N01-GM-6-2119 from the National Institute of General Medical Sciences.     

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

Chromosome 7 short arm deletion and craniosynostosis a 7p-syndrome

Summary A patient with craniosynostosis and a small deletion of part of the short arm of chromosome 7 is described. A review of the literature indicates that craniosynostosis has occurred in at least four of the five infants (the fifth having microcephaly) affected by structural changes (resulting in deletion) within the terminal region of the short arm of chromosome 7.Supported by: National Foundation March of Dimes Grant CA-90, National Institutes of Health Grants, No. 5S01 RR05655-07 and No. P01 GM 15 253-08     

Computer simulation of diffusely-connected neuronal populations

This paper describes computer simulations of diffusely-connected neuronal populations. Main findings are that diffuse monosynaptic linkages between populations are selectively sensitive to synchronized clusters of action potentials in the pre-synaptic population; that diffusely-connected excitatory recurrent collaterals tend to produce rhythmic series of synchronized clusters; and that diffusely-connected inhibition (both recurrent and afferent) tend to reduce the number of cells participating in a given synchronized cluster and thereby the overall transfer rate. However, recurrent inhibition tends to increase the rate at which synchronized clusters are produced by recurrent excitation. These results suggest the speculation that diffusely connected neuronal populations are particularly prone to deal with synchronized clusters of action potentials.This work has been supported by Grant GB 33687 of the National Science Foundation, Grant 1-R01-NS-10781-01 COM of the National Institutes of Health, and by a fellowship from Zonta, International     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

In vitro maturation of Arbacia punctulata oocytes and initiation of heavy body formation

Summary An ultrastructural study of in vitro maturation of A. punctulata oocytes was undertaken to determine when heavy body formation was initiated. No heavy bodies were seen in germinal vesicle oocytes or in oocytes undergoing germinal vesicle breakdown or polar body formation. Heavy bodies were only observed in ova examined one to two hours after pronuclear formation. Several small heavy bodies were seen in sections of eggs fixed as early as three hours after the pronucleus had formed. The number of these structures in the egg cytoplasm increased with time. Therefore it is concluded that heavy body formation in sea urchin ova is a phenomenon following nuclear maturation.Contribution No. 222 from the Institute for Molecular and Cellular Evolution. Supported in part by grants from the National Institutes of Health (5-T01-HD00026-09 to the Fertilization and Gamete Physiology Training Program at the Marine Biological Laboratory and predoctoral fellowship 1-F01-GM-36,719-01A1 to C. M. Conway) and the National Science Foundation (GB3899 to C. B. Metz).The authors are grateful to Drs. A. F. Conway, Giovanni Giudice, and Gertrude W. Hinsch for consultation and criticism of this work.     

Magnetic field measurements near RF induction heaters

We discuss initial magnetic field strength measurements made around radiofrequency (RF) induction heaters. These measurements were made with a monitor developed for the National Institute for Occupational Safety and Health (NIOSH) by the National Bureau of Standards (NBS). The monitor has a dynamic range of .01 to 10,000 A2/m2, a frequency range of 300 kHz to 100 MHz, an isotropic response (+/- .3 dB) with three mutally orthogonal loop antennas that have the ability to measure and display each of three orthogonal magnetic field components, a high probe burnout protection level of 20,000 A2/m2, and an accuracy of +/- 1.0 dB at 13 calibration frequencies. The portable survey monitor was used to measure the magnetic field strengths in the vicinity of 16 RF induction heaters. Typically these induction heaters are operated continuously for several hours. The maximum field strengths (without duty factor correction) ranged from 15 to 4,500 A2/m2 and were measured 5 to 51 cm from the loop applicators of the induction heaters. At locations commonly occupied by workers (ie, approximately 30 to 76 cm from heaters), the fields ranged from .01 to 300 A2/m2 (without duty factor correction).     

Spike timing dependent synaptic plasticity in biological systems

 Association of a presynaptic spike with a postsynaptic spike can lead to changes in synaptic efficacy that are highly dependent on the relative timing of the pre- and postsynaptic spikes. Different synapses show varying forms of such spike-timing dependent learning rules. This review describes these different rules, the cellular mechanisms that may be responsible for them, and the computational consequences of these rules for information processing and storage in the nervous system. Received: 16 January 2002 / Accepted: 3 June 2002 Acknowledgements. This research is supported in part by a National Science Foundation grant IBN 98-08887 (awarded to PDR), and by National Institutes of Health grants R01-MH49792 (awarded to CCB), R01-MH60996 (awarded to CCB), and R01-MH60996 (awarded to PDR). Correspondence to: P. D. Roberts (e-mail: robertpa@ohsu.edu)     

Fine structure of the pterin layer in the dermis of Rana pipiens

Summary The fine structure of the pterin layer was investigated in both wild type Rana pipiens and Rana pipiens homozygous for the speckle mutant gene. No difference in morphology of the layer was noted between the wild type and mutant. The layer lines the outer surface of the stratum compactum of the dermis and separates this stratum from the stratum spongiosum. The pterin layer consists of extra-cellular material and contains membrane-bounded granules filled with fine spicules. Many of the spicules are somewhat similar in appearance to the initial calcification loci present in developing membrane bone. The layer first appears in the tadpole at approximately stage 14 (Taylor and Kollros, 1946); subsequent developmental stages are described.This work was supported by United States Public Health Service Fellowships (to G.E.W.) 1-FO2-CA32869-01 and 5-FO2-CA32869-02; (to L.W.B.) 1-FO2-GM-32, 906-01 and 1-FO2-GM-32-906-02; by funds from an Institutional Grant to the University of Colorado from the American Cancer Society (L.W.B.); by National Research Council (Canada) Grant # A6209 (L.W.B.); by Program Project HD-02282 of the National Institutes of Health; and by Health Sciences Advancement Award FR-02084 from the National Institutes of Health.     

Dynamical synaptic plasticity: a model and connection to some experiments

 Using a modified version of a phenomenological model for the dynamics of synaptic plasticity, we examine some recent experiments of Wu et al. [(2001) J Physiol 533:745–755]. We show that the model is quantitatively consistent with their experimental protocols producing long-term potentiation (LTP) and long-term depression (LTD) in slice preparations of rat hippocampus. We also predict the outcome of similar experiments using different frequencies and depolarization levels than reported in their results. Received: 3 September 2002 / Accepted in revised form: 22 October 2002 / Published online: 24 February 2003 Correspondence to: H.D.I. Abarbanel (e-mail: hdia@jacobi.ucsd.edu) Acknowledgements. We are very grateful to A. Selverston and D. Feldman for conversations about this work. This work was partially supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Engineering and Geosciences, under grants No. DE-FG03-90ER14138 and No. DE-FG03-96ER14592, by a grant from the National Science Foundation, NSF PHY0097134, by a grant from the Army Research Office, DAAD19-01-1-0026, by a grant from the Office of Naval Research, N00014-00-1-0181, and by a grant from the National Institutes of Health, NIH R01 NS40110-01A2. This work was also partially supported by M. Ciencia y Tecnologa BFI2000-0157 (R.H.).     

Replication and differentiation in vitro of epidermal cells from normal human skin and from benign (psoriasis) and malignant (basal cell cancer) hyperplasia

Summary The normal steady state in human epidermis reflects a balance between the rates of cell proliferation and of cell differentiation (keratinization). In certain diseases, such as psoriasis and basal cell cancer, increased proliferative activity is associated with abnormalities in keratinization. These events have been thought to be directly related. However, studies in which the epithelial cells from normal epidermis, psoriasis, and basal cell cancer were grown in vitro suggest that abnormalities of the keratinization process do not directluy result from hyperproliferation. Alterations in keratinization probably result from abnormal dermal-epidermal interactions, independent of any dermal effects on mitosis. This work was supported by grants from the National Cancer Institute (1 PO 1 CA 11536) and the National Institute of Arthritis and Metabolic Disease (1 PO 1 AM 1551-01).     

Systemic lupus erythematosus and C1q: A quantitative ELISA for determining C1q levels in serum

The authors would like to acknowledge the National Institutes of Health for funding (grant number 5R01AR053734).     

Culture of adult rat lung cells: Benzo(a)pyrene metabolism and mutagenesis

Summary A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2×10⁸ viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies. Visiting scientist from Hungary. This research was supported by Grant 5 R01 CA20022 and Public Health Service Contract N01 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Nuclear membrane contributions to the Golgi complex

Summary Electron microscopic examination of a variety of rapidly growing or differentiating mammalian and avian cells suggests that many of the Golgi vesicles and saccules arise directly from the outer nuclear membrane. Evidence for this interpretation includes: (1) the presence of a continuum of vesicles which appears to originate from the outer nuclear membrane and to enlarge gradually into saccules in the region of the Golgi membrane complex; (2) the absence of ribosomes on the nuclear blebs and the vesicles formed in these regions along the nuclear envelope; (3) the presence of active nuclear vesiculation near the Golgi region in cells essentially devoid of rough and/or smooth endoplasmic reticulum, and (4) the demonstration of peroxidase activity in the cisternae of the nuclear envelope and in vesicles extending in rows from the nuclear envelope to the Golgi complex. Supported by Research Grants HE 04061-09 (HEM), C-5315 and 1S01 FR-05109-01, Project 10 from the National Institutes of Health, Bethesda, Maryland.     

Detection of individual human chromosomes by chromosome in situ suppression hybridization using PCR-amplified bacteriophage library probes

We used the polymerase chain reaction (PCR) to prepare chromosome-specific probes from the bacteriophage λ library LA01NS01, prepared at the Los Alamos National Laboratory from flow sorted human chromosome 1. By using oligonucleotide primers flanking the EcoRI insertion site of the Charon 21A vector, we were able to amplify the human sequences preferentially in the library up to 9.1 kb (maximum insert size). The product of the PCR reaction was nick translated with incorporation of biotinylated residues and used with fluorescence in situ hybridization to observe metaphase chromosomes by fluorescence microscopy. This technique allows for a relatively easy method for preparation of chromosome-specific library probes for “chromosome painting.” The quality of the results obtained by this method compares favorably to those obtained by using bulk-purified library inserts. This method offers potential advantages in terms of cost and east of use.     

The importance of stroma in morphogenesis and functional activity of urogenital epithelium

Summary Urogenital morphogenesis and cytodifferentiation are presented in the context of the epithelial-stromal interaction. The essential role of stroma in these processes is reviewed. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. The study was supported in part by Grant No. PDT-8 from the American Cancer Society, and Contract Grants N01-CP-55649 and N01-CP-75875 from the National Cancer Institute.     

Constrained sequence alignment

This paper presents a dynamic programming algorithm for aligning two sequeces when the alignment is constrained to lie between two arbitrary boundary lines in the dynamic programming matrix. For affine gap penalties, the algorithm requires onlyO(F) computation time andO(M+N) space, whereF is the area of the feasible region andM andN are the sequence lengths. The result extends to concave gap penalties, with somewhat increased time and space bounds. K.-M. C. and W. M. were supported in part by grant R01 LM05110 from the National Library of Medicine. R. C. H. was supported by PHS grant R01 DK27635.     

Endogenous HPRT activity in mycoplasmas isolated from cell cultures

Summary Five mycoplasma species most frequently isolated from cell cultures were tested for the presence of endogenous hypoxanthine phosphoribosyl-transferase (HPRT), activity. All of the five, cultured in cell-free medium, contained variable but significant levels of HPRT. Two strains ofM. hyorhinis exhibited a 13-fold difference in their specific HPRT activity. When infected with any of these mycoplasma species, HPRT-deficient mouse cell mutants rapidly acquired a cell-associated HPRT activity; however, the cells remained sensitive to HAT medium and resistant to 6-thioguanine. On the other hand, normal HPRT-positive cells deliberately infected with the mycoplasmas uniformly became sensitive to HAT medium. The apparent transfer of mycoplasma-specific HPRT activity to HPRT-deficient cells may be used as a sensitive measure of cell infection by these mycoplasma strains. The HPRT activities of mycoplasmas share several common properties so that they can be distinguished easily from the mammalian HPRT isozymes. Compared to the animal cell enzymes, the mycoplasmal HPRT activities are less heat stable, more strongly inhibited by 6-thioguanine, and in general migrate more slowly in electrophoresis at a neutral pH. This work was supported in part by PHS Research Grants 5 R01 GM21014 and 1 P03 GM19100 (Genetics Center Grant to Albert Einstein College of Medicine), and PHS Research Contracts N01 GM 6-2119 and N01-AG-4-2865 (to the Institute for Medical Research), from the National Institute of General Medical Sciences and National Institute on Aging. S. S. is a recipient of a Faculty Research Award from the American Cancer Society.