[Physical and chemical properties of the condensate of breathed-out air in healthy people]. / Fiziko-khimicheskie svoistva kondensata vydykhaemogo vozdukha u zdorovykh liudei.

The physical-and-chemical condition of condensate of breathed-out moisture (CBM) was studied by the computer-aided analysis of axis-symmetric drops' form in healthy subjects; such condition was found to depend on sex, age and smoking-addiction of the examined persons. The surface tension of the mentioned moisture correlated with its viscoelastic index and with relaxation. The metabolites of nitric oxide, lipids, urea, lactic acid (not uric acid) and of hydrogen peroxide were found to influence the physical-and-chemical parameters of respiratory moisture. Hydrogen peroxide had a reverse correlation with the CBM viscoelastic module. A status of the pulmonary surfactant can be evaluated by using the studied physical-and-chemical CBM parameters.

The two PDGF receptors maintain conserved signaling in vivo despite divergent embryological functions.

Gene targeting studies have indicated that the two receptors for PDGF, alpha and beta, direct unique functions during development. Distinct ligand affinities, patterns of gene expression, and/or mechanisms of signal relay may account for functional specificity of the two PDGF receptor isoforms. To distinguish between these factors, we have created two complementary lines of knockin mice in which the intracellular signaling domains of one PDGFR have been removed and replaced by those of the other PDGFR. While both lines demonstrated substantial rescue of normal development, substitution of the PDGFbetaR signaling domains with those of the PDGFalphaR resulted in varying degrees of vascular disease. This observation provides a framework for discussing the evolution of receptor tyrosine kinase functional specificity.

A novel transgenic marker for migrating limb muscle precursors and for vascular smooth muscle cells.

A unique pattern of LacZ expression was found in a transgenic mouse line, likely due to regulatory elements at the site of integration. Two new genes flanking the transgene were identified. At early stages of development, the transgene is transiently expressed in ventro-lateral demomyotomal cells migrating from the somites into the limb buds. At late developmental stages and in the adult, lacZ staining marks vascular smooth muscle cells throughout the vascular bed, with the exception of the major elastic arteries, and in pericytes. No expression was detected in skeletal and smooth muscles. Different patterns of expression in vascular smooth muscles was observed at distinct levels of the vascular tree, in arteries as well as in veins. Vessel injury, resulting in stimulation of smooth muscle cells proliferation and migration, is associated with transgene down-regulation. After the formation of neointima thickening, it is reactivated. This transgenic insertion may therefore be used as a useful marker to identify novel physiological cues or genetic elements involved in the regulation of the vascular smooth muscle phenotype(s). It may also provide an experimental tool for studying vasculature and the involvement of pericytes in regulating microvascular homeostasis.

Different impact of p53 and p21 on the radiation response of mouse tissues.

Mammalian tissues differ dramatically in their sensitivity to genotoxic stress, although the mechanisms determining these differences remain largely unknown. To analyse the role of p53 and p21 in determination of tissue specificity to DNA damage in vivo, we compared the effects of gamma radiation on DNA synthesis on whole-body sections of wild type, p53-deficient and p21-deficient mice. A dramatic reduction in 14C-thymidine incorporation after gamma irradiation was observed in the majority of rapidly proliferating tissues of wild type and p21-/- but not in p53-/- mice, confirming the key role of p53 in determination of tissue response to genotoxic stress in vivo and suggesting that p53-mediated inhibition of DNA synthesis does not depend on p21. Rapid radiation induced p53-dependent apoptosis was mapped to the areas of high levels of p53 mRNA in radiation sensitive tissues analysed (white pulp in the spleen and bases of crypts in small intestine), indicating that p53 regulation at the mRNA level is a determinant of cellular sensitivity to genotoxic stress. High p53 mRNA expression is inherited as a recessive trait in cell-cell hybrids suggesting the involvement of a negative control mechanism in the regulation of p53 gene expression.

In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with beta-lactoglobulin and whey acidic protein promoters.

The expression pattern of human serum albumin (HSA) in transgenic mice carrying various HSA genomic sequences driven either by the mouse whey acidic protein (WAP) or the sheep beta-lactoglobulin (BLG) promoters, was compared. The pattern of HSA expression in both WAP/HSA and BLG/HSA transgenic lines was copy number independent, and the major site of ectopic expression was the skeletal muscle. Although an equal proportion of expressors was determined in both sets of mice (approximately 25% secreting >0.1 mg/ml), the highest level of HSA secreted into the milk in the WAP/HSA transgenic lines was one order of magnitude lower than in the BLG/HSA lines. Despite this difference, the HSA expression patterns in the mammary gland were similar and consisted of two levels of variegated expression. Studies using mammary explant cultures revealed a comparable responsiveness to the lactogenic hormones insulin, hydrocortisone, and prolactin, although the WAP/HSA gene constructs were more sensitive to the hydrocortisone effect than were the BLG/HSA vectors. When HSA vectors were stably transfected into the mouse mammary cell line CID-9, they displayed a hierarchy of expression, dependent upon the specific complement of HSA introns included. Nevertheless, the expression of HSA in four out of five WAP/HSA constructs was similar to their BLG/HSA counterparts. This construct-dependent, and promoter-independent, hierarchy was also found following transfection into the newly established Golda-1 ovine mammary epithelial cell line.

Development of avian tibial dyschondroplasia: gene expression and protein synthesis.

Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion-which was significantly enlarged-was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy.

Beta-lactoglobulin/human serum albumin fusion genes do not respond accurately to signals from the extracellular matrix in mammary epithelial cells from transgenic mice.

Mammary epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously, we demonstrated that non-HSA-secreting transgenic strains retain the potential to express the HSA transgene in vitro and that mammary epithelial cell cultures from non-HSA-secreting strains express higher levels of HSA when grown on tissue culture plastic than they do when grown on collagen. In this study we studied the expression of BLG/HSA fusion genes in epithelial cell cultures of additional transgenic strains and additional substrata. Our results show that: (1) The BLG/HSA fusion gene in only one of seven HSA-secreting or nonsecreting transgenic strains tested accurately responded to signals from the EHS matrix; (2) HSA DNA sequences dominantly affected the activity of BLG as well as the whey acidic protein promoters; and (3) HGF/SF induced both milk proteins and HSA gene expression. These results suggest that the response to the extra cellular matrix (ECM) plays a key role in the expression of BLG/HSA fusion genes and that the function of the regulatory elements within the promoter regions of milk protein genes involved in response to the ECM, in developmental and in tissue specificity, very much depend on the downstream gene sequences.

Dual regulation of beta-lactoglobulin/human serum albumin gene expression by the extracellular matrix in mammary cells from transgenic mice.

Mammary explants and epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously we demonstrated that mammary explants from virgin female transgenic mice synthesize and secrete high levels of HSA during the first day in culture. Here we present a detailed analysis of endogenous and transgene expression during the first 20 h of mammary explant cultures. We show that HSA genes as well as endogenous milk protein genes are rapidly induced upon explantation. Unexpectedly, HSA was synthesized also in mammary explants from strains that do not secrete HSA into the milk, indicating the existence of a cryptic potential to express the transgene. Histological examination revealed that some luminal epithelial cells detached from the underlying extracellular matrix (ECM) soon after explantation. Epithelial cell cultures from nonsecreting strains grown on plastic rapidly induced transgene expression and secreted higher levels of HSA into the medium compared to cells grown on collagen. These results suggest that tissue organization and most likely the interaction of epithelial cells with the ECM are intimately involved in the control of HSA transgene expression.

The distal human myoD enhancer sequences direct unique muscle-specific patterns of lacZ expression during mouse development.

Transgenic mice carrying the bacterial lacZ reporter gene under the control of the regulatory elements of the human myoD gene have been produced. The developmental expression of the myoD reporter transgene in somites, limb buds, visceral arches, and cephalocervical regions was studied in transgenic embryos by beta-gal staining. In somites, the spatiotemporal pattern of transgene expression was different from other muscle-specific regulatory and structural genes and revealed that myoD-expressing cells arise in distinct patterns in somites that are dependent on position along the anterior-posterior (AP) body axis (occipital and cervical vs thoracic and more posterior myotomes). Transgene expression did not follow a strict anterior to posterior sequence of activation and therefore was not strictly correlated with somite developmental age. Moreover, the pattern of transgene expression along the dorsal-ventral myotomal axis was dependent on somite position along the anterior-posterior axis. While myoD expression is first detected after the myotome is well-formed, transgene expression in the dorsal and ventral medial lips of the dermatome suggests a function for myoD in the expansion of the myotome. Whole-mount in situ hybridization confirmed that these unique patterns of transgene expression in somites, as well as expression in limb buds, visceral arches, and other myogenic centers, are concordant with the distribution of endogenous myoD transcripts. These results shed new light on the developmental differences between myotomes at different positions along the AP and DV axis and demonstrate a unique axial pattern of somitic myoD expression, suggesting a specific role of myoD in myotome lineage determination and differentiation.

Quail myoD is regulated by a complex array of cis-acting control sequences.

Quail myoD (QmyoD) is the earliest myoD family member expressed in quail somites and its transcription is initiated in response to early developmental signals. We have investigated the transcriptional regulation of QmyoD to define the cis-acting sequences required for tissue-specific and correct developmental expression. The QmyoD gene locus was isolated and sequenced and its regulatory properties were characterized. We identified three distinct regions of cis-acting regulatory sequences that control the expression of reporter gene constructs following DNA transfection into cell lines and cultured primary quail cells. The first, a complex distal control region (DCR), 11.5 kb upstream of the gene, contains three separable enhancer activities. Two of these DCR enhancer activities are tissue specific and can be autoactivated. In addition, these same two enhancers and the entire DCR direct somite- and muscle-specific expression of a reporter gene in transgenic mice. Sequence analysis of the DCR enhancers reveals clusters of E-boxes, MEF2 binding motifs, and the stretches of sequence identity with the human myoD enhancer. Second, the promoter region has sequences which act positively to direct expression in both muscle and nonmuscle cells as well as sequences that repress expression specifically in nonmuscle cells. The third control region, the PR, is located -3.3 to -5 kb from the transcription start site and directs muscle-specific expression in cultured cells. This analysis demonstrates that QmyoD has multiple control regions and that some features of myoD regulation are conserved between mammals and birds.

Dramatic heterogeneity of transgene expression in the mammary gland of lactating mice: a model system to study the synthetic activity of mammary epithelial cells.

We studied the expression of human serum albumin (HSA) driven by the ovine beta-lactoglobulin promoter in the mammary glands of lactating mice from five independent transgenic strains, by employing combined in situ hybridization and immunostaining techniques. Four strains displayed a heterogeneous pattern of expression: mice of strains 91 and 92 expressed the transgene in only a fraction of the lobules, whereas in strains 69 and 83 all lobules contained cells expressing HSA. In all four strains the patterns of expression within expressing lobules corresponded to the morphology of alveolar cells and to the extent of local milk secretion, suggesting that filling of alveolus with secreted material was accompanied by asynchronous downregulation of transgene expression. In situ hybridization to the endogenous milk protein genes alpha-lactalbumin, beta-casein, and whey acidic protein revealed a uniform pattern of expression in lactating mammary glands of transgeneic and in four out of five non-transgeneic mice. In the fifth control mouse, we detected downregulation of gene expression in lobules containing alveoli distended by secreted milk. The pattern of expression of the three endogenous genes was greatly disturbed after a short (3-hr) unilateral closure of mammary glands, and very much resembled the pattern of expression of the HSA transgenes. These results demonstrate that transgeneic mice provide a useful model to study the factors that regulate the synthetic activity of mammary epithelial cells.

Embryonic activation of the myoD gene is regulated by a highly conserved distal control element.

MyoD belongs to a small family of basic helix-loop-helix transcription factors implicated in skeletal muscle lineage determination and differentiation. Previously, we identified a transcriptional enhancer that regulates the embryonic expression of the human myoD gene. This enhancer had been localized to a 4 kb fragment located 18 to 22 kb upstream of the myoD transcriptional start site. We now present a molecular characterization of this enhancer. Transgenic and transfection analyses localize the myoD enhancer to a core sequence of 258 bp. In transgenic mice, this enhancer directs expression of a lacZ reporter gene to skeletal muscle compartments in a spatiotemporal pattern indistinguishable from the normal myoD expression domain, and distinct from expression patterns reported for the other myogenic factors. In contrast to the myoD promoter, the myoD enhancer shows striking conservation between humans and mice both in its sequence and its distal position. Furthermore, a myoD enhancer/heterologous promoter construct exhibits muscle-specific expression in transgenic mice, demonstrating that the myoD promoter is dispensable for myoD activation. With the exception of E-boxes, the myoD enhancer has no apparent sequence similarity with regulatory regions of other characterized muscle-specific structural or regulatory genes. Mutation of these E-boxes, however, does not affect the pattern of lacZ transgene expression, suggesting that myoD activation in the embryo is E-box-independent. DNase I protection assays reveal multiple nuclear protein binding sites in the core enhancer, although none are strictly muscle-specific. Interestingly, extracts from myoblasts and 10T1/2 fibroblasts yield identical protection profiles, indicating a similar complement of enhancer-binding factors in muscle and this non-muscle cell type. However, a clear difference exists between myoblasts and 10T1/2 cells (and other non-muscle cell types) in the chromatin structure of the chromosomal myoD core enhancer, suggesting that the myoD enhancer is repressed by epigenetic mechanisms in 10T1/2 cells. These data indicate that myoD activation is regulated at multiple levels by mechanisms that are distinct from those controlling other characterized muscle-specific genes.

Synthesis and secretion of caseins by the mouse mammary gland: production and characterization of new polyclonal antibodies.

Polyclonal antibodies to mouse alpha- and beta/gamma-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both alpha- and beta/gamma-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse alpha-lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of gamma-casein in the milk, the amount of this protein compared to alpha- or beta-caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.

Ectopic expression of beta-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation and in situ localization.

We produced transgenic mice carrying the native sheep beta-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains. In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.

Synthesis and secretion of human serum albumin by mammary gland explants of virgin and lactating transgenic mice.

Transgenic mice were produced, carrying hybrid genes comprised of the ovine beta-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences. In situ hybridization revealed high levels of BLG/HSA hybrid mRNA, confined to the epithelial cells of the lactating mammary gland with a several hundred fold lower concentration in virgin mammary glands. During the first 24 h in culture, exceptionally high levels of HSA were secreted from explants of virgin mice, independent of hormonal control. HSA secretion was reduced considerably during subsequent days in culture and became dependent on the presence of insulin, hydrocortisone and prolactin. This temporal and hormonal pattern of regulation of HSA was different than that found for the secretion of caseins. In contrast to the vast difference in the mRNA content, the amount of HSA secreted from explants derived from lactating mice during the first 24 h in culture was only 2- to 5-fold higher than that found with explants from virgin transgenic mice, suggesting post-transcriptional control of HSA synthesis. The high-level synthesis and secretion of HSA in mammary explants of lactating mice was also dependent on the presence of insulin, hydrocortisone and prolactin. This study confirms previous suggestion that mammary explants from virgin transgenics may serve as a powerful tool for screening the potential of transgenic animals to secrete foreign proteins in their milk.

The expression of the regulatory myosin light chain 2 gene during mouse embryogenesis.

The fast skeletal muscle myosin light chain 2 (MLC2) gene is expressed specifically in skeletal muscles of newborn and adult mice, and has no detectable sequence homology with any of the other MLC genes including the slow cardiac MLC2 gene. The expression of the fast skeletal muscle MLC2 gene during early mouse embryogenesis was studied by in situ hybridization. Serial sections of embryos from 8.5 to 12.5 days post coitum (d.p.c.) were hybridized to MLC2 cRNA and to probes for the myogenic regulatory genes MyoD1 and myogenin. The results revealed different temporal and spatial patterns of hybridization for different muscle groups. MLC2 transcripts were first detected 9.5 d.p.c. in the myotomal regions of rostral somites, already expressing myogenin. Surprisingly, at the same stage, a weak MLC2 signal was also detected in the cardiomyocytes. The cardiac expression was transient and could not be detected at later stages while the myotomal signal persisted and spread to the more caudal somites, very similar to the expression of myogenin. Beginning from 10.5 d.p.c., several extramyotomal premuscle cells masses have been demarcated by MyoD1 expression. MLC2 transcripts were detected in only one of these cell masses. Although, transcripts of myogenin were detected in all these cell masses, the number of expressing cells was significantly lower than that observed for MyoD1. By 11.5 d.p.c., all three hybridization signals colocalized in most extramyotomal muscle-forming regions, with the exception of the diaphragm and the hindlimb buds, where only few cells expressed MLC2 and more cells expressed MyoD1 than myogenin. At 12.5 d.p.c., all three studied genes displayed a similar spatial pattern of expression in most muscle-forming regions. However, in some muscles, the MyoD1 signal spread over more cells compared to myogenin or MLC2. Our results are consistent with the suggestion that multiple myogenic programs exist for myoblasts differentiating in the myotome and extramyotomal regions.

Ectopic expression of MyoD1 in mice causes prenatal lethalities.

A variety of differentiated cell types can be converted to skeletal muscle following transfection with the myogenic regulatory gene MyoD1. To determine whether MyoD1 is a dominant muscle regulator in vivo, mouse fertilized eggs were microinjected with a beta-actin/MyoD1 gene. Ectopic expression of MyoD1 during mouse embryogenesis led to embryonic lethalities, the cause of which is not known. Transgenic embryos died before midgestation. The majority of tested embryos between 7.5 and 9.5 days, although retarded compared to control littermates, differentiated normally into tissues representative of all three germ layers. In most transgenic embryos there was no indication of myogenic conversion. The expression of the introduced gene was detected in all ectodermal and mesodermal tissues but was absent in all endodermal cells. Forced expression of MyoD1 was associated with the activation of myogenin and MLC2 (but not myf5 or MRF4) genes in non-muscle cell types, demonstrating the dominant regulatory function of MyoD1 during development. These results demonstrate that ectopic MyoD1 expression and activation of myogenin and MLC2 have no significant effects in the determination of cell lineages or the developmental fate of differentiated mesodermal and ectodermal cell lineages.