Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.
...
PMID:Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface. 32 10

The incubation in vitro of plasmid pBR322 DNA with glucose 6-phosphate (Glc-6-P) has been shown to have a mutagenic effect when the plasmid was transformed into wild-type Escherichia coli. To further investigate the modifications of DNA by the reducing sugar Glc-6-P, we have developed an in vivo model to monitor plasmid DNA mutations. E. coli strains that are defective for phosphoglucose isomerase (strain DF40) alone or phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (strain DF2000) accumulate Glc-6-P when grown in gluconate minimal medium in the presence of glucose. These strains and the control strain K10 were transformed with pAM006, a plasmid that carries the genes for ampicillin resistance and beta-galactosidase production, and grown for 24 hr under conditions that prompted the accumulation of Glc-6-P. An increase in plasmid mutations was observed (7- and 13-fold) that was associated with the increased intracellular levels of Glc-6-P (20- and 30-fold) present in the DF40 and DF2000 E. coli strains, respectively. Growth of the mutant bacteria in gluconate minimal medium does not increase the intracellular levels of Glc-6-P or the rate of plasmid mutations over background. Further characterization of the mutated plasmid DNA showed that insertions, deletions, and point mutations were responsible for the loss of beta-galactosidase production. The increase in plasmid mutations as a function of increased intracellular Glc-6-P levels suggests that the accumulation of adducts formed by Glc-6-P and other reducing sugars may contribute to DNA damage.
...
PMID:Elevated glucose 6-phosphate levels are associated with plasmid mutations in vivo. 282 85

As the major proteins of adult keratinocytes, keratins provide biochemical markers for exploring mouse epidermal embryogenesis. Here, we used a modified method of whole-mount in situ hybridization to track skin-specific expression of endogenous keratin mRNAs throughout embryogenesis. To monitor transcriptional regulation, we coupled this with beta-galactosidase expression of a human epidermal keratin promoter-driven transgene. These studies have radically changed our perception of how the program of gene expression becomes established during epidermal development. Specifically, we have discovered that (1) basal keratin (K5 and K14) genes are first detected at E9.5 in a highly regional fashion, and surprisingly as early as the single layered ectodermal stage; (2) the early patterns do not correlate with morphogenesis per se, but rather with regional variations in the embryonic origin of underlying mesenchyme, supporting morphogenetic criteria that early inductive cues are mesenchymal; (3) epidermal keratin genes are expressed in periderm, supporting the notion that this layer arises from ectodermal stratification, even though it is simple epithelial-like in morphology and is subsequently sloughed during development; (4) later embryonic patterns of K5 and K14 gene expression parallel proliferative capacity and not stratification; and (5) K1 and K10 mRNAs are first detected as early as E13.5, and their patterns correlate with differentiation and not stratification. These patterns of epidermal gene expression led us to explore whether potential transcriptional regulators of these genes are expressed similarly. We show that AP2 (but not Sp1) cRNAs hybridize in a pattern similar to, but preceding that of basal keratin cRNAs. Finally, using gene expression in cultured cells, we demonstrate that AP2 has a strong inductive effect on basal keratin expression in a cellular environment that does not normally possess AP2 activity.
...
PMID:Programming gene expression in developing epidermis. 752 78

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.
...
PMID:Sustainability of keratinocyte gene transfer and cell survival in vivo. 919 11

Although there are several methods for introducing the genes to keratinocytes in vivo, expression of transgene does not last long enough for effective keratinocyte gene therapy. In this study, we added bovine papilloma virus 1 (BPV) DNA into expression vectors with the lacZ gene driven by metallothionein and keratin 10 promoters, and we transferred them into keratinocytes in vivo using the naked DNA method, and measured beta-gal activity in keratinocytes. The results showed that beta-galactosidase activity of vectors with the BPV DNA was clearly higher than that without the DNA. Moreover, time-course experiment disclosed that the activity of the BPV vector declined at a lower rate than that of the control vector, suggesting this fragment prolonged transgene expression. These results should prove useful for understanding gene regulation in keratinocytes in vivo and for developing potential expression vectors for keratinocyte gene therapy.
...
PMID:Expression vector with DNA of bovine papilloma virus 1 for keratinocyte gene therapy. 1080 28

The replication kinetics and cytological changes of normal human oral keratinocytes (NHOK) isolated from the basal surface of oral epithelial sheet and cultured as dispersed cells in low (0.15 mM) Ca(2+) medium without serum were analyzed. Replicating NHOK were quantitated by cell count and identified by [(3)H]thymidine uptake. Cell morphology was analyzed by phase contrast and transmission electron microscopy, and by cytochemical staining for endogenous beta-galactosidase (beta-gal) activity, involucrin, and cytokeratin types 1 and 10 (K1/K10). Primary NHOK obtained from 15 different donors whose ages ranged from 21 to 62 years consistently showed three distinct phases of replication, i.e., exponential, senescing, and senescent, which were independent of the donors' age. Initially, the cells replicated exponentially for a period of 20 days with a doubling time of 26.6 +/- 3.5 h. They then gradually entered replication arrest over a period of 18 days. The cells underwent a maximum of 22.1 +/- 2.8 population doublings. The onset of gradual replication arrest coincided with an increase in the fraction of cells, which stopped DNA synthesis within a maximum of 48 h and which stained for beta-gal. The fraction of terminally differentiated cells stained for K1/K10 did not increase until nearly all the cells had stopped replicating (senescent phase) and maximal beta-gal staining had been reached. Subsequently, the percentage of beta-gal stained cells actually decreased while the percentage of those stained for K1/K10 increased to a maximum of 80-90% within 2-3 weeks. Exposure of exponentially replicating NHOK to 5-aza-2'-deoxycytidine (5-aza CdR) inhibited DNA replication within 18-48 h and induced terminal differentiation 6 days later. In contrast, exposure of these cells to 1.5 mM Ca(2+) induced expression of involucrin and K1/K10 within 48 h without inhibiting DNA synthesis. Thus, replication arrest preceded differentiation in NHOK serially subcultured in vitro; however, differentiation could be induced without replication arrest.
...
PMID:In vitro replication and differentiation of normal human oral keratinocytes. 1089 80

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.
...
PMID:Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli. 1107 68

In Drosophila, the establishment of dorsoventral polarity of the developing embryo depends on the expression of at least 11 maternally acting genes. Mutant females that lack any of these gene activities produce normally shaped eggs that develop into dorsalized embryos. The female sterile K10 mutation differs from these mutants, because in addition to the dorsalized development of the embryo, it causes a dorsalization of the egg shape. During oogenesis, the K10 gene is specifically expressed in the oocyte. Antibodies raised against a beta-galactosidase-K10 fusion protein were used to visualize the K10 product in ovaries by indirect immunofluorescence. The protein, which contains a putative DNA recognition helix, accumulates in the nucleus of the oocyte, where it is assumed to have a regulatory function. Our results thus indicate that the controlled expression of some of the genes of the oocyte nucleus is essential for the determination of the dorsoventral polarity of the oocyte and possibility of the developing embryo.
...
PMID:Role of the oocyte nucleus in determination of the dorsoventral polarity of Drosophila as revealed by molecular analysis of the K10 gene. 1255 95

1. By digitonin lysis of penicillin spheroplasts of Escherichia coli a particulate fraction P(1) was previously obtained that supported the sustained synthesis of alkaline phosphatase when supplied with amino acids, nucleotide triphosphates and other cofactors. This P(1) fraction, when subjected to mild ultrasonic treatment in the presence of sucrose and Mg(2+), yielded the P(1)(S) fraction, consisting of integrated particulate subcellular particles containing DNA and RNA. 2. The P(1)(S) fraction from E. coli K10 wild type (R(+) (1)R(+) (2)P(+)) grown under repressed conditions supported the immediate synthesis of alkaline phosphatase in vitro. The synthesis occurred in phases. The first was followed by a lag, and then there was a linear rapid phase that continued for at least 3hr. Actinomycin D inhibited the appearance of the second phase. It was concluded that the particles are programmed to synthesize enzyme even when prepared from repressed cells, and therefore that synthesis of the specific messenger RNA for alkaline phosphatase in vivo was not inhibited when the bacteria were grown in an excess of inorganic phosphate. 3. Phosphate inhibited synthesis of enzyme to the same extent with the P(1)(S) fractions of two constitutive strains as with the P(1)(S) fraction of the wild-type strain. 4. Inorganic phosphate inhibited amino acid incorporation with the P(1)(S) fraction and also inhibited enzyme synthesis in vitro. The effect on amino acid incorporation could be partially overcome by adding Mn(2+) to the incubation mixtures. However, Mn(2+) inhibited the synthesis of alkaline phosphatase. Also, inhibition of the incorporation of [(32)P]CTP into RNA was overcome by Mn(2+). The effect of phosphate on amino acid uptake was most probably due to a phosphorolysis of RNA by polynucleotide phosphorylase, also present in the P(1)(S) fraction. This phosphorolysis may be responsible for the instability of messenger RNA in vitro and in vivo. 5. Phosphate also specifically inhibited the formation of alkaline phosphatase, since it did not affect markedly the induced formation of beta-galactosidase by the same P(1)(S) fraction. The specific effect is attributed to the prevention of formation of the enzymically active dimer from precursors, a Zn(2+)-dependent reaction. It is suggested that the repression of the synthesis of alkaline phosphatase in vivo in the wild-type strain was the sum of these two effects.
...
PMID:THE BIOSYNTHESIS OF ALKALINE PHOSPHATASE WITH A PARTICULATE FRACTION OF ESCHERICHIA COLI. 1433 60

p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and DeltaN. DeltaNp63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, DeltaN- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. DeltaNp63alpha declines with Ca2+-induced differentiation, while a smaller DeltaN-form, DeltaNp63s, persists, suggesting unique functions of the two DeltaN-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, DeltaNp63alpha, DeltaNp63p40, TAp63alpha, TAp63gamma or beta-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT-PCR and western blot analyses revealed that overexpression of DeltaNp63p40, which lacks the entire alpha-tail present in DeltaNp63alpha, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length DeltaNp63alpha, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the alpha-tail of DeltaNp63alpha in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the alpha-terminus requires the cooperation of the DeltaN domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The DeltaN-terminus is sufficient to maintain keratinocytes in a proliferative state, as both DeltaN forms block Ca2+-mediated p21WAF1 induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63alpha or TAp63gamma. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.
...
PMID:Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis. 1608 16


1

---