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Biomedical subjects

H Green

Publications and source records attributed to H Green.

At least 109 records · Page 6Linked to original sources
PubMed1988

Suppression of SV40-promoted gene expression by differentiation of preadipose cells.

When a plasmid bearing the chloramphenicol acetyltransferase (CAT) gene under the control of an SV40 early promoter is introduced into preadipose or adipose cells of line 3T3-F442A, the promoter directs high levels of transient expression of CAT. However, when the plasmid is introduced into preadipose cells and the cells are then allowed to differentiate into adipose cells, the expression of the CAT gene is suppressed. In this process, the plasmid is not changed detectably in amount, topology, or state of methylation. Stably transformed preadipose cells bearing an integrated plasmid express the transferase, but if the cells are allowed to differentiate, the expression of the gene is similarly suppressed. The decline in CAT activity is associated with a decrease in the transcription rate of the gene. Transcription of a gene coding for neomycin phosphotransferase driven by the SV40 promoter is also greatly diminished by differentiation. Because suppression of CAT does not occur when the gene is under control of a retroviral long terminal repeat (LTR), a specific mechanism exists for the recognition and inactivation of the SV40 early promoter during differentiation.

Adipose Tissue↗
PubMed1988

New techniques for the grafting of cultured human epidermal cells onto athymic animals.

Cultures of human epidermal cells may be used to generate epidermis on athymic recipients. We describe two novel techniques for grafting such cultures. Both techniques permit the generation of typical human epidermis within 7 d. Both techniques result in less graft contraction than conventional grafting, and there is no difficulty in distinguishing the human epidermis generated by the graft from the epidermis of the recipient animal. Starting with a single human biopsy, epidermis may be generated on a great many experimental animals; such grafts should therefore provide uniform material for investigation of the properties of human epidermis.

Animals↗
PubMed1988

Oxidative potential in developing rat diaphragm, EDL, and soleus muscle fibers.

To examine the effect of postnatal development on changes in oxidative potential of fibers of specific types (I, IIa, IIb, and IIc) in the rat diaphragm, determinations of succinate dehydrogenase (SDH) activity were made using microphotometric measures of optical density. Samples of the costal region of the diaphragm were extracted from 56 male Wistar rats ranging in age from 8 to 85 days and subgrouped into seven developmental periods (1, 2, 3, 4, 6, 9, and 12 wk). For type I fibers, increases of 17% (P less than 0.05) in SDH activity occurred during 2nd wk, remained elevated through 4th wk, and increased further (P less than 0.05) to 137% of 1-wk values by the end of 6th wk. No further increases were noted between 6 and 12 wk. A similar maturational trend was evident for type IIa fibers, although SDH activities remained higher throughout development when compared with type I fibers. In contrast, SDH in type IIb fibers, although increasing by 14% during the first two measurement weeks (P less than 0.05), declined from 6 to 9 wk before ultimately reaching a value similar to 3 wk. SDH activity was also assessed in a typical slow- (soleus) and fast-twitch (extensor digitorum longus, EDL) muscle of the hindlimb to contrast their development with that of the diaphragm. Generally, SDH in type I and IIa fibers was approximately 40 and 20% higher, respectively, in the diaphragm than in matched fiber types in the other muscles throughout development (diaphragm greater than EDL greater than soleus).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1987

Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-alpha and epidermal growth factor.

In common methods of cell cultivation, multiplication takes place in cells distributed uniformly or in small colonies and the number of cells increases exponentially. In contrast, an isolated colony of coherent epidermal keratinocytes, as it grows larger, departs drastically from exponential growth, and instead increases its radius at a constant rate over time. The rate of increase of colony radius is 8-fold greater in the presence of epidermal growth factor (EGF) and 10-fold greater in the presence of transforming growth factor-alpha (TGF-alpha): the resulting megacolonies may become 30-50 times greater in area and cell number than colonies grown in the absence of the growth factors. Growth of a colony depends on outward migration of the rapidly proliferating cells located in a thin rim close to the colony perimeter. The effect of EGF and TGF-alpha in promoting multiplication must depend on their ability to increase the rate of this cell migration.

Cell Division↗
PubMed1987

Expression of an exogenous growth hormone gene by transplantable human epidermal cells.

Retrovirus-mediated gene transfer was used to introduce a recombinant human growth hormone gene into cultured human keratinocytes. The transduced keratinocytes secreted biologically active growth hormone into the culture medium. When grafted as an epithelial sheet onto athymic mice, these cultured keratinocytes reconstituted an epidermis that was similar in appearance to that resulting from normal cells, but from which human growth hormone could be extracted. Transduced epidermal cells may prove to be a general vehicle for the delivery of gene products by means of grafting.

DNA, Recombinant↗
PubMed1987

Cytotoxicity and mutagenicity of low intensity, 248 and 193 nm excimer laser radiation in mammalian cells.

The cytotoxicity of 193 and 248 nm excimer laser radiation was compared to that produced by a germicidal lamp (predominantly 254 nm) using Chinese hamster ovary cells (CHO), and a human diploid fibroblast line, AG-1522A. Excimer laser radiation at 248 nm (3.5 X 10(2) w/m2) and germicidal radiation (5.3 X 10(-5) w/m2) caused toxicity in both cell lines, with the AG-1522A cells (D37 = 7-8 J/m2) being slightly more sensitive than the CHO cells (D37 = 11 J/m2). Incident 193 nm radiation was less cytotoxic than 248 nm to AG-1522A and CHO cells with D37 values of 18 and 85 J/m2, respectively. The mutagenic potential of UV excimer radiation at 193 and 248 nm was evaluated using the hypoxanthine guanine phosphoribosyl transfer assay system with CHO cells. Excimer laser radiation at 248 nm induced mutation in proportion to dose (1.7 X 10(-5) resistant colonies per survivor per J/m2 incident radiation) up to 14 J/m2, similar to results reported for 254 nm light. However, excimer laser radiation at 193 nm did not cause mutation greater than the dark control. The decreased cytotoxicity and mutagenicity of 193 nm radiation may be due to the shielding of the nucleus by cytoplasmic and membrane components or to the formation of different DNA photoproducts. These differences between 193 and 248 nm radiation may be important in choosing an excimer wavelength for ablation in biological systems.

Animals↗
PubMed1987

Three clonal types of keratinocyte with different capacities for multiplication.

Colony-forming human epidermal cells are heterogeneous in their capacity for sustained growth. Once a clone has been derived from a single cell, its growth potential can be estimated from the colony types resulting from a single plating, and the clone can be assigned to one of three classes. The holoclone has the greatest reproductive capacity: under standard conditions, fewer than 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate. The paraclone contains exclusively cells with a short replicative lifespan (not more than 15 cell generations), after which they uniformly abort and terminally differentiate. The third type of clone, the meroclone, contains a mixture of cells of different growth potential and is a transitional stage between the holoclone and the paraclone. The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones.

Cell Division↗
PubMed1987

Potential methodologic problems with in vivo immunoneutralization of pancreatic polypeptide.

Dogs with chronic pancreatic fistulae were given 0.5 ml of nonimmune rabbit serum or antibody S5, an antibody raised against the C-terminal pancreatic polypeptide (PP) hexapeptide. A 3-h infusion of secretin (125 ng/kg/h) and CCK8 (50 ng/kg/h) was started 30 min after injecting serum. Exogenous BPP (400 pmol/kg/h) was administered during the middle secretin/CCK hour. In a second protocol, 30 min after injecting nonimmune serum or PP-anti-serum, the animals were fed 15 g/kg cooked ground beef. Pretreatment with S5 enhanced secretin/CCK-induced bicarbonate outputs; protein outputs did not differ. Exogenous BPP inhibited pancreatic secretion, even in S5-treated animals. Meal-induced pancreatic secretion was not altered by S5 pretreatment. Significant increments in PP were measured by radioimmunoassay during administration of secretin/CCK and during BPP infusion. Anti-PP pretreatment abolished the former and significantly decreased, but did not abolish, the latter. The meal evoked significant postprandial increments in PP which were essentially abolished following S5 pretreatment. A physiological role for PP cannot be proved or refuted because antiserum pretreatment failed to block the effects of exogenous hormone. The latter must be established before excluding a peptide's physiological role based on negative in vivo immunoneutralization data.

Animals↗
PubMed1987

The reorganization of microtubules and microfilaments in differentiating keratinocytes.

Using immunofluorescence techniques, we have examined the microtubules and microfilaments in colonies of terminally differentiating human keratinocytes in tissue culture. The undifferentiated keratinocytes contained numerous microtubules, which radiated from a centrosomal organization center (MTOC). Differentiating keratinocytes, which leave the basal layer and begin to synthesize involucrin, displayed an altered cytoskeleton. Thick mats and coils of microtubules formed throughout the cytoplasm of the differentiated squames, and microfilaments were no longer visible after staining with phalloidin. Instead, only scattered stipples of phalloidin-stained material were observed. The results suggest that the terminal differentiation of epidermal cells involves a reorganization not only of the keratin filaments but of the entire cytoskeleton.

Actin Cytoskeleton↗
PubMed1986

Structure and evolution of the human involucrin gene.

Involucrin is a keratinocyte protein that first appears in the cell cytosol, but ultimately becomes cross-linked to membrane proteins by transglutaminase. The gene for human involucrin has now been cloned and sequenced. The central segment of the coding region contains 39 repeats of a 30 nucleotide sequence whose ten encoded amino acids include three glutamines and two glutamic acids. This segment must have originated by successive duplications. Later duplications of modified sequences within the central segment can also be identified. Flanking the central segment lie shorter coding segments, a part of which must have given rise to the central segment. The flanking segments also show homology to a simpler 30 nucleotide sequence from which they likely originated. The evolution of involucrin as a substrate of transglutaminase and an envelope precursor was evidently made possible by this process of repeated mutation and duplication.

Amino Acid Sequence↗
PubMed1986

The nucleotide sequence of three genes participating in the adipose differentiation of 3T3 cells.

When 3T3 cells undergo adipose differentiation, they are reprogrammed by changes in gene expression of sufficient magnitude to greatly alter the protein composition of the cells. Three participating genes encode glycerophosphate dehydrogenase, a lipid-binding protein, and a serine protease. These three genes have now been cloned and sequenced. Their exon/intron structures are described, together with some interesting peculiarities and some regions of common sequence. It remains to be demonstrated whether the information for common participation of the genes in the program of differentiation resides in controlling elements within the regions sequenced.

Adipose Tissue↗
PubMed1986

The generation of insulin-like growth factor-1--sensitive cells by growth hormone action.

Insulin-like growth factor-1 (IGF-1), a mitogenic polypeptide, is usually considered the sole effector by means of which growth hormone increases tissue mass. However, growth hormone, but not IGF-1, directly promotes the differentiation of cultured preadipocytes to adipocytes. Adipocytes newly differentiated from precursor cells in response to growth hormone were shown to be much more sensitive to the mitogenic effect of IGF-1 than the precursor cells. The result of IGF-1 action is therefore a selective multiplication of young differentiated cells (cloned expansion). This supports the concept of a dual effector system in which the preferred target cells of IGF-1 action are created by the direct action of growth hormone.

Adipose Tissue↗
PubMed1986

Involucrin acts as a transglutaminase substrate at multiple sites.

Involucrin is a keratinocyte protein with a specialized function in terminal differentiation. Synthesized initially as a soluble protein, it later becomes a preferred substrate for a membrane-bound transglutaminase and becomes cross-linked into an insoluble envelope. When a crude keratinocyte extract containing about 2% involucrin is heated to 95 degrees, most proteins precipitate, but all of the involucrin remains in solution, where it is over 90% pure. This step has been incorporated into a simplified procedure for purification of the protein. Like intact involucrin, polypeptide fragments formed by the tryptic hydrolysis of involucrin are good substrates for the keratinocyte transglutaminase. Evidently amino acid residues participating in the enzyme-catalyzed cross-linking are distributed at numerous sites along the involucrin molecule.

Binding Sites↗
PubMed1985

The activation of specific gene transcription in the adipose conversion of 3T3 cells.

During the adipose conversion of 3T3 cells, there occur sequential changes in cellular protein and mRNA composition. To determine if there are also changes in transcription, we have studied the transcripts of specific genes in isolated nuclei prepared before and after adipose conversion. Transcription of three genes encoding adipocyte-specific proteins was detectable only in adipocytes, whereas transcription of actin and collagen type I genes occurred in both adipocytes and preadipocytes. The activation of transcription of adipocyte-specific genes was not synchronous. Thus temporal differences in the appearance of different adipocyte mRNAs probably result from differences in the times of activation of transcription.

Adipose Tissue↗
PubMed1985

Enzymatic cross-linking of involucrin and other proteins by keratinocyte particulates in vitro.

A transglutaminase-catalyzed cross-linking process characteristic of keratinocytes leads to the formation of the insoluble corneocyte envelope. The essentials of this process take place in vitro in a reconstituted system derived from subcellular fractions. A particulate fraction containing membrane-bound envelope precursor proteins and the enzyme transglutaminase is combined with cytosolic proteins; when the enzyme is activated by Ca++, cytosolic proteins are removed from solution and cross-linked to particulate proteins. This interaction is cell-type-specific, since particulates derived from fibroblasts and also containing transglutaminase activity cannot substitute for those of keratinocytes. Involucrin, a cytosolic protein known to be a precursor of the envelope, is more efficiently cross-linked than other cytosolic proteins. The cross-linking of proteins of the particulate fraction (membrane proteins) is promoted by the presence of involucrin.

Acyltransferases↗
PubMed1985

Cell size as a determinant of the clone-forming ability of human keratinocytes.

Keratinocytes isolated from human epidermis and subsequently cultured may form clones if they are 11 micron or less in diameter but are irreversibly committed to further enlargement and terminal differentiation if they are 12 micron or more in diameter. When a founding cell of 11 micron or less forms a small rapidly growing clone in culture, the cells of that clone are able to found new colonies even when their diameter is as great as 20 micron. As the clone becomes larger and grows more slowly, the maximal size of its clonogenic cells is reduced toward that of the epidermis. A cultured cell of up to 20 micron in diameter can, when it divides, give rise to clonogenic progeny smaller than itself, thus reversing the process of enlargement. Cells larger than 20 micron cannot divide and therefore cannot be rescued from terminal differentiation. It is concluded that when keratinocytes multiply rapidly, they extend reversibly the maximal size at which they are capable of generating clones into the range usually characteristic of terminally differentiating cells. It is proposed that this mechanism enables the keratinocyte to accommodate an increased rate of multiplication to its need to attain a large size during terminal differentiation.

Cell Division↗