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Biomedical subjects

J M Clark

Publications and source records attributed to J M Clark.

At least 199 records · Page 11Linked to original sources
PubMed1985

Freeze-fracture observations on mammalian oocytes.

Freeze-fracture studies on mammalian oocytes have been hampered by the relatively small numbers of cells available at a given time as well as by difficulties encountered in effectively freezing these large, watery cells. We have nevertheless pursued this area because of the benefits of visualizing membrane faces involved in various fusion reactions by the freeze-fracture method. Our observations indicate no overall change in intramembranous particle (IMP) distribution before and after sperm penetration, although the question of possible alterations of these structures at the precise locus of sperm attachment remains open. Preliminary statistical analysis indicates that there is a much higher IMP density on the P face than on the E face of the plasma membrane and that the microvillar membranes bear more IMPs than those of the intermicrovillus regions. Probes of lipid subclasses were used to determine the distribution of cholesterol and anionic lipid in the egg plasma membrane. Filipin and tomatin showed extensive complex formation in microvillus as well as nonmicrovillus regions, whereas anionic lipids (using polymyxin B) have been difficult to detect on the oocyte surface. These results are discussed relative to current views of membrane fusion mechanisms.

Animals↗
PubMed1985

Regulation of discoidin I gene expression in dictyostelium discoideum by cell-cell contact and cAMP.

We have previously presented evidence that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression in D discoideum [Berger EA, Clark JM: Proc Natl Acad Sci USA 80:4983, 1983]. Here we provide genetic evidence to support this hypothesis by examining gene expression in a cohesion-defective mutant, strain EB-21, which enters the developmental program but is blocked at the loose mound stage. When this strain was developed in suspension, the cells remained almost entirely as single amoebae, unlike the wild type, which formed large multicellular aggregates. In both strains, discoidin I mRNA levels were low in vegetative cells but rose sharply during the first few hours of development. However, the peak level reached at 8 hr in EB-21 exceeded that observed in wild type, and while the level declined markedly over the next few hours in wild type, it remained highly elevated in the mutant. Thus, there was a correlation between the inability of EB-21 to form normal cell-cell contacts and its deficiency in inactivating discoidin I gene expression. Previous studies from several laboratories, including this one, have demonstrated that exogenously added cAMP can block or reverse the changes in gene expression normally seen upon cell disaggregation. This has led us to propose that cAMP serves as a second messenger regulating the expression of contact-regulated genes. Here we provide additional support for this hypothesis. Intracellular cAMP levels rapidly dropped several-fold when wild type tight cell aggregates were disaggregated and remained low as the cells were cultured in the disaggregated state. Furthermore, overexpression of discoidin I mRNA late in development in EB-21 was corrected by addition of high concentrations of cAMP. These results are consistent with a second messenger function for cAMP in the contact-mediated regulatory response, and they indicate that the cAMP response machinery for discoidin I gene expression is capable of functioning in the cohesion-defective EB-21 strain.

Cyclic AMP↗
PubMed1985

The organization of collagen in cryofractured rabbit articular cartilage: a scanning electron microscopic study.

Adult rabbit articular cartilage was prepared for scanning electron microscopy using, in order, glutaraldehyde fixation, enzymatic removal of proteoglycan, dehydration in ethanol, cryofracture in liquid nitrogen, and critical-point drying. Enzymes were effective in fixed material. Fixation, cryofracture, alignment of fracture surfaces with "split lines," and retention of subchondral bone were found to be necessary steps for the preservation of collagen detail. The fibrous framework was found to be similar to that proposed by Benninghoff and favored by more recent phase-contrast microscopic studies. Vertical fibers extending from subchondral bone and a network of tangentially oriented superficial fibrils converge in the transitional zone. No random layer is seen. Pericellular capsules interdigitate with the vertical fibers. When cartilage is prepared in a manner that minimizes tissue damage, scanning electron microscopy provides useful, unique information.

Animals↗
PubMed1985

Auditory and vestibular function in hyperbaric oxygen.

The therapeutic value of high partial pressures of oxygen is limited by the toxicity of oxygen. Pulmonary damage, visual impairment, and convulsions are known hazards during hyperoxic exposure, but dose-effect relationships have not been quantified for specific organs or functions. As part of an integrated study of organ oxygen tolerance in man (Predictive Study V of the Institute for Environmental Medicine), auditory and vestibular function was evaluated in man before, during, and after exposure to 100% oxygen at 3 atmospheres absolute pressure for 2 to 3.5 hours. No alterations in hearing or equilibrium were detected, suggesting that ototoxicity is not a factor that would limit exposure under these conditions.

Adult↗
PubMed1984

Eucaryotic initiation factor 4B of wheat germ binds to the translation initiation region of a messenger ribonucleic acid.

Purified preparations of eucaryotic initiation factor 4B (eIF4B) from wheat germ bind the monocistronic, uncapped, mRNA satellite tobacco necrosis virus RNA (STNV RNA) in nitrocellulose-mediated binding assays. This reaction is mRNA specific and yields dissociation constants (Kd) in the 10(-7)-10(-8) M range, depending upon the particular enzyme preparation tested. Purified wheat germ eIF4A, in the presence or absence of ATP, does not bind STNV RNA efficiently, but added eIF4A and ATP do enhance the efficiency of the eIF4B-dependent binding of STNV RNA. Wheat germ eIF4B binds the oligonucleotide containing the 5'-terminal 52 nucleotides of STNV RNA (designated 1-52) with the same affinity as intact STNV RNA. This binding affinity is less with the 1-44 oligonucleotide of STNV RNA and does not occur with the 1-33 oligonucleotide of STNV RNA that contains the 5'-terminal untranslated region and the initiator AUG codon at positions 30-32 of this mRNA. Wheat germ eIF4B therefore binds the translation initiation region of STNV RNA, and this binding requires up to 20 nucleotides on the 3' side of the initiator AUG codon of this mRNA. Wheat germ eIF4B also efficiently binds an oligonucleotide containing nucleotides from positions 13-52 in from the 5' terminus of STNV RNA, thereby establishing that the postulated 5'-terminal stem and loop secondary structure of STNV RNA [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] is not functional or essential for this specific binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Eukaryotic Initiation Factors↗
PubMed1984

Alternative surfaces for microcarrier culture of animal cells.

As part of an effort to broaden the applicability and efficiency of microcarrier cell culture various alternative new microcarriers were synthesized. The microcarriers were compared as substrates for the growth of several types of cells and with respect to binding of proteins from the culture medium. Cross-linked dextran has been found to be the most suitable material for a microcarrier matrix and was used as the matrix for the new microcarriers. One type of microcarrier was synthesized so that the charges necessary for cell attachment were present only in the surface layer of the microcarrier in the form of N,N,N-trimethyl-2- hydroxyaminopropyl groups. The resulting microcarriers had a very low capacity to bind proteins from the culture medium (e.g. albumin and IgG) and such proteins could be removed from the cultures more efficiently than when using previous microcarriers. A new principle was used for the development of the other type of microcarrier. A surface layer of cross-linked denatured collagen provided the surface for growth of cells. These microcarriers provided a "natural" substrate for cell growth and allowed improved attachment and spreading of cells with epithelial morphology. Harvesting cells from these microcarriers with proteolytic enzymes was more efficient than with previous microcarriers. The two alternative microcarriers should result in improved process efficiency and have a potential value in the preparation of live vaccines.

Animals↗
PubMed1984

Interacting effects of hypoxia adaptation and acute hypercapnia on oxygen tolerance in rats.

To define the effects of hypoxia adaptation on tolerance to a useful range of O2 pressures, groups of 20 or more rats were exposed to O2 at 1.0, 1.5, 2.0, 3.0, and 4.0 ATA before and after adaptation to an inspired PO2 (PIO2) of 71 Torr for 5 days. Effects of acute hypercapnia on O2 tolerance in hypoxia-adapted rats were also determined by exposing rats to the same O2 pressures with an inspired PCO2 (PICO2) of 60 Torr. In nonadapted rats exposed to O2 at 1.0, 1.5, 2.0, 3.0, and 4.0 ATA, 50% mortality (LD50) occurred at 76.4, 26.8, 17.4, 9.0, and 6.4 h, respectively. LD50 values in O2-CO2 at the same pressures were 77.1, 24.5, 15.6, 3.4, and 1.7 h. Hypoxia-adapted rats had only 20% mortality in O2 at 1.0 ATA, and survivors were killed at 336 h. In O2-CO2 at 1.0 ATA, mortality was 85% with an LD50 at 282 h. LD50 values in hypoxia-adapted rats at O2 pressures of 1.5, 2.0, 3.0, and 4.0 ATA were 63.1, 22.5, 7.9, and 3.8 h, respectively. Corresponding values in O2-CO2 were 29.5, 18.7, 4.9, and 1.9 h. Exposure to O2 at 4.0 ATA caused nearly immediate onset of violent convulsions in hypoxia-adapted rats compared with a 50% incidence of convulsions at 3.2 h in nonadapted rats. These data indicate that hypoxia-adaptation increases pulmonary O2 tolerance but reduces central nervous system (CNS) O2 tolerance. However, the enhanced pulmonary O2 tolerance in hypoxia-adapted rats is greatly diminished when acute hypercapnia is superimposed on O2 exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1983

Specific cell-cell contact serves as the developmental signal to deactivate discoidin I gene expression in Dictyostelium discoideum.

Specific cell-cell contact is a major regulatory signal controlling cell differentiation in Dictyostelium discoideum, causing dramatic changes in the developmental program of gene expression. In this report, we focus on the relationships between specific cell-cell contact and the activity of the genes for discoidin I, an endogenous lectin that has been implicated in the cell-cell cohesion process. By performing quantitative RNA dot-hybridization assays and RNA gel blot-hybridization analyses, using as a probe a recombinant plasmid containing a discoidin I cDNA insert, we have measured changes in discoiding I mRNA levels during normal development and in response to specific manipulations of the state of cellular aggregation. Our major findings are as follows. (i) During normal development on filters, there is a close temporal correspondence between the establishment of specific cell-cell contacts and the decline in discoidin I mRNA levels. By the tight-aggregate stage, discoidin I mRNA is barely detectable. (ii) When tight aggregates are disaggregated and the cells are maintained in the disaggregated state, there is a dramatic rise in discoidin I mRNA content. (iii) When cells are developed in suspension (conditions that interfere with the establishment of tight cell-cell contacts), discoidin I mRNA accumulates to abnormally high levels, and these persist well after the levels in filter-developed cells have declined. Taken together, these results strongly suggest that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression; thus, a contact-deactivated gene for which a recombinant DNA probe is available has now been identified. Furthermore, we demonstrate that exogenous cAMP almost completely blocks the disaggregation-induced reactivation of discoidin I gene expression. Possible mechanistic relationships between specific cell-cell contact, intracellular cAMP levels, and developmental gene expression are discussed.

Journal Article↗
PubMed1983

Gastric emptying following premedication with glycopyrrolate or atropine.

A double-blind study of the effects of glycopyrrolate or atropine i.v. on gastric emptying was undertaken in 30 non-pregnant and 30 pregnant women. Gastric emptying, which was assessed by the uptake of orally administered paracetamol, was decreased in the patients in the first trimester of pregnancy when compared with the non-pregnant patients. Following the administration of glycopyrrolate, gastric emptying was decreased in the non-pregnant patients and decreased further in the pregnant patients.

Acetaminophen↗
PubMed1983

Human respiration at rest in rapid compression and at high pressures and gas densities.

Ventilation (V), end-tidal PCO2 (PACO2), and CO2 elimination rate were measured in men at rest breathing CO2-free gas over the pressure range 1-50 ATA and the gas density range 0.4-25 g/l, during slow and rapid compressions, at stable elevated ambient pressures and during slow decompressions in several phases of Predictive Studies III-1971 and Predictive Studies IV-1975. Inspired O2 was at or near natural O2 levels during compressions and at stable high pressures; it was 0.5 ATA during decompressions. Rapid compressions to high pressures did not impair respiratory homeostasis. Progressive increase in pulmonary gas flow resistance due to elevation of ambient pressure and inspired gas density to the He-O2 equivalent of 5,000 feet of seawater was not observed to progressively decrease resting V, or to progressively increase resting PACO2. Rather, a complex pattern of change in PACO2 was seen. As both ambient pressure and pulmonary gas flow resistance were progressively raised, PACO2 at first increased, went through a maximum, and then declined towards values near the 1 ATA level. It is suggested that this pattern of PACO2 change results from interaction on ventilation of 1) increase in pulmonary resistance due to elevation of gas density with 2) increase in respiratory drive postulated as due to generalized CNS excitation associated with exposure to high hydrostatic pressure. There may be a similar interaction between increased gas flow resistance and increase in respiratory drive related to nitrogen partial pressure and the narcosis resulting therefrom.

Adult↗
PubMed1983

Breathing patterns during submaximal and maximal exercise in elite oarsmen.

Continuous breath-by-breath measurements of ventilatory parameters were performed during submaximal and maximal treadmill exercise in 21 highly conditioned oarsmen. Average maximum values of O2 uptake, minute ventilation (VI), tidal volume (VT), and respiratory frequency (f) were 6.60 l/min (73.5 ml X kg-1 X min-1), 200 l/min, 3.29 l, and 62 breaths/min, respectively. During the transition from moderate to heavy submaximal exercise, VT and f increased progressively. At near-maximal to maximal work loads, VT plateaued and then decreased slightly, while f continued to increase. Increase in f at the start of exercise was achieved predominantly by an abrupt decrease in expiratory duration (TE) with an equally abrupt, but much smaller, decrease in inspiratory duration (TI). During the transition from submaximal to maximal exercise, both TE and TI decreased progressively. Although f appeared to be entrained by stepping rate in a few subjects, the dominant trend during submaximal to maximal exercise was characterized by a relatively small increase in stepping rate with a much larger increment in f. Our data are consistent with the conclusion that exercise breathing patterns are determined by many interacting factors that vary at different work loads, in different individuals, and are probably also influenced by physical conditioning and previous experience.

Adult↗
PubMed1983

The separation of harvested cells from microcarriers: a comparison of methods.

Two techniques are described which have been designed to separate harvested cells from microcarriers. The requirements of efficient recovery and high viability of the cells are met by both procedures. Differences both in size and density between cells and microcarriers allow separations which are based on differential centrifugation or filtration. After trypsinizing the cells from the microcarriers, separation was performed by either ; a) low-speed centrifugation on Ficoll-Paque or, b) filtration through nylon meshes. The methodologies for both techniques are presented. Up to 78% of the total cells were recovered by discontinuous gradient centrifugation using Ficoll-Paque. similar results were obtained when separating Vero, BS-C-1 and MRC-5 cells from Cytodex 1, Cytodex 2 and Cytodex 3 microcarriers. Equally high recoveries were obtained by filtration through an 88 micron pore size nylon mesh. Data are presented for the separation of both Vero and HeLa cells from all 3 types of microcarriers after filtration through 53 and/or 88 micron meshes. Greater than 92% viability of the recovered cells was consistently obtained and their growth properties upon subsequent reculturing were unaffected by either separation procedure. Differential sedimentation by unit gravity provides adequate cell recovery for many applications, but yields are significantly increased by using one or other of the methods described here. Both techniques are rapid and efficient. In addition, differential centrifugation provides a concentrated suspension of the recovered cells, while the nylon meshes for filtration are reusable and can be autoclaved at least 5 times.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

A new method to characterize the translation initiation sites of messenger ribonucleic acids.

RNA can be radiolabeled in vitro with 125I in uridine and cytidine residues by initial mercuration of these pyrimidine residues by the method of Dale et al. [Dale, R. M. K., Martin, E., Livingston, D. C., & Ward, D. C. (1975) Biochemistry 14, 2447-2457] and subsequent electrophilic displacement of the mercury with 125I+ generated by the method of Commerford [Commerford, S. L. (1971) Biochemistry 10, 1993-2000]. These two reactions can be manipulated to produce intact high specific activity 125I-labeled mRNA containing approximately equal specific activity in their 5-[125I]iodouridine and 5-[125I]iodocytidine residues. In vitro radiolabeling of satellite tobacco necrosis virus RNA (STNV RNA) by this procedure yields a 125I-labeled mRNA that is biologically active in ribosome protection analyses in that one obtains the correct translation initiation fragments of the mRNA. Exhaustive digestion of a specific 125I-labeled 32 nucleotide long initiation fragment of STNV RNA using four separate nucleotide-specific digestion (hydrolysis) reactions yields four different populations of 125I-labeled digestion products that can be resolved by two-dimensional fingerprint procedures. Characterization of all these 125I-labeled digestion products followed by overlap nucleotide sequence comparisons of these specific digestion products allows a nucleotide sequence determination of the original 32 nucleotide long translation initiation fragment of this mRNA. This suggests that this overall in vitro procedure can be used to determine the nucleotide sequence of the translation initiation site(s) of any mRNA.

Base Sequence↗