Use of anti-infective agents during lactation: Part 2--Aminoglycosides, macrolides, quinolones, sulfonamides, trimethoprim, tetracyclines, chloramphenicol, clindamycin, and metronidazole.

Because many antibiotics are excreted into breast milk, it can be difficult for a practitioner to choose an antibiotic for a lactating patient that will have minimal risks to her nursing infant. This article is the second of a three-part series discussing the use of anti-infective agents during lactation. The authors review general information regarding use and common side effects for several classes of antibiotics. They also summarize information, including documented milk concentrations, milk-to-plasma ratios, and other pharmacokinetic properties, in a table that can help practitioners choose antibiotics that may be considered safe to use in the lactating mother.

Expression and regulation of type I BMP receptors during early avian sympathetic ganglion development.

We have investigated the expression and regulation of the mRNAs for the type I BMP receptors, BMPR-IA and BMPR-IB, in quail embryos in vivo and in neural crest cultures in vitro. BMPR-IB mRNA was expressed in the primordial sympathetic ganglia at stage 17, soon after the first expression of Cash-1 mRNA, the avian homolog of the Drosophila transcription factor achaete-scute. BMP-4 mRNA was detected in the dorsal aorta at stage 17, coincident with BMPR-IB mRNA expression in the sympathetic ganglia. BMPR-IA mRNA was first expressed in the sympathetic ganglia at stage 18. Moreover, BMP-4 ligand mRNA was detected in the sympathetic ganglia starting at stage 18. BMPR-IA and BMPR-IB were differentially regulated in cultured neural crest cells. BMPR-IB was expressed in primary outgrowths of neural crest cells but was downregulated after primary outgrowths were harvested and replated in secondary cultures. In secondary cultures of neural crest cells, exogenous BMP-2 and BMP-4 increased the expression of BMPR-IA but decreased the expression of BMPR-IB. The expression of both type I BMP receptors was inhibited by exogenous TGF-beta1. Our results suggest distinct roles for BMPR-IA and BMPR-IB in the development of the sympathoadrenal phenotype from cells of the neural crest.

Use of anti-infective agents during lactation: Part 1--Beta-lactam antibiotics, vancomycin, quinupristin-dalfopristin, and linezolid.

Because many antibiotics are excreted into the breast milk, it can be difficult for a practitioner to choose an antibiotic for a lactating patient that will have minimal risks to her nursing infant. This article is the first of a three-part series discussing the use of anti-infective agents during lactation. The authors review general information regarding use and common side effects of several classes of antibiotics. They also summarize information, including documented milk concentrations, milk-to-plasma ratios, and other pharmacokinetic properties, in a table that can help practitioners choose antibiotics that may be considered safe for the lactating mother.

Bone morphogenetic proteins-2 and -4 attenuate apoptosis in a cerebellar primitive neuroectodermal tumor cell line.

Similarities between primitive neuroectodermal tumors and central nervous system (CNS) progenitor cells have evoked interest in the response of these tumors to endogenous growth factors. The bone morphogenetic proteins (BMPs) have recently been found to regulate survival and differentiation of CNS progenitor cell populations. In this study, we investigated the effects of BMP-2, BMP-4, and BMP-6 on the undifferentiated cerebellar primitive neuroectodermal tumor or medulloblastoma cell line DAOY. Analysis by reverse transcriptase-polymerase chain reaction showed that mRNAs for type IA and type II BMP receptors were present in control cultures. In cultures treated with BMP-2, mRNAs for BMP receptor type IB and the activin R-I receptor became evident. Cultures were analyzed for total cell counts, proliferating cell nuclear antigen (PCNA), and apoptotic DNA fragmentation. There was a significant increase in total cell number in the BMP-2 and BMP-4 treatment groups, without any change in PCNA reactivity, and a dramatic decrease in the proportion of apoptotic nuclei at concentrations of BMP-2 and BMP-4 above 5 ng/ml (P<0.001). These effects were not observed with BMP-6, TGF-beta1 or GDNF. These results suggest that the increase in total cell number is due to the attenuation of apoptosis by BMP-2 and BMP-4. The anti-apoptotic effect of BMP-2 and BMP-4 on this neuroectodermal cell line has potential clinical implications for neuroectodermal tumors.

Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures.

Previous work has demonstrated that the bone morphogenetic proteins (BMP)-2, BMP-4, and BMP-7 can promote the development of tyrosine hydroxylase (TH)-positive and catecholamine-positive cells in quail trunk neural crest cultures. In the present work, we showed that mRNA for the type I bone morphogenetic protein receptor IA (BMPR-IA) was present in neural crest cells grown in the absence or presence of BMP-4. We have used a replication-competent avian retrovirus to express a constitutively active form of BMPR-IA in neural crest cells in culture. Cultures grown in the absence of BMP-4 and infected with retrovirus containing a construct encoding this activated BMPR-IA developed five times more TH-immunoreactive and catecholamine-positive cells than uninfected control cultures or cultures infected with virus bearing the wild-type BMPR-IA cDNA. The number of TH-positive cells which developed was dependent on the concentration of virus bearing the activated receptor cDNA used in the experiments. Most TH-positive cells which developed also contained viral p19 protein. Total cell number was not affected by infection with the virus containing the activated receptor construct. The effect of the activated receptor was phenotype-specific since infection with the virus bearing the activated receptor cDNA did not alter the number or morphology of microtubule-associated protein (MAP)2-immunoreactive cells, which are distinct from the TH-positive cell population. These findings are consistent with the observation that MAP2-positive cells are not affected by the presence of BMP-4. Taken together, these results suggest that activity of BMPR-IA is an important element in promoting the development of the adrenergic phenotype in neural crest cultures.

Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Encapsidated conformation of bacteriophage T7 DNA.

The structural organization of encapsidated T7 DNA was investigated by cryo-electron microscopy and image processing. A tail-deletion mutant was found to present two preferred views of phage heads: views along the axis through the capsid vertex where the connector protein resides and via which DNA is packaged; and side views perpendicular to this axis. The resulting images reveal striking patterns of concentric rings in axial views, and punctate arrays in side views. As corroborated by computer modeling, these data establish that the T7 chromosome is spooled around this axis in approximately six coaxial shells in a quasi-crystalline packing, possibly guided by the core complex on the inner surface of the connector.

In vivo footprinting with limiting amounts of embryo tissues: a role for C/EBP beta in early hepatic development.

Regulatory factors important for the developmental control of genes have been identified by genetic studies or by examining the ontological expression profiles of proteins that were originally characterized in adult tissues; direct biochemical studies of transcription factors within small amounts of embryo tissues have been limited. We have found that the ligation-mediated PCR (LM-PCR) technique can detect specific dimethylsulfate modifications in genomic DNA from as few as several thousand cells, making it technically feasible to identify protein-DNA interactions in pools of nascent embryo tissues. Herein we show that LM-PCR can reveal methylation protections on the albumin gene enhancer in embryonic mouse hepatocytes, indicating occupancy of a C/EBP factor binding site. Comparison of the in vivo protection pattern with that obtained from the in vitro analysis of different C/EBP isoforms suggests that in embryonic hepatocytes, C/EBP beta is bound to the albumin gene enhancer. Detailed protocols are provided so that the approach can be used to study other genes in developing embryos.

Nucleosome positioning properties of the albumin transcriptional enhancer.

Considering the importance of nucleosome position with regard to how regulatory factors recognize their binding sites in chromatin, we have investigated the inherent nucleosome positioning properties of a transcriptional enhancer of the albumin gene. In the liver, where the albumin gene is highly expressed, the enhancer exists in an array of precisely positioned, nucleosome-like particles with transcription factors bound. In the absence of specific binding factors, such as in non-liver tissues or in polynucleosome arrays assembled in vitro, nucleosomes are randomly positioned over the enhancer. Herein we investigate the intrinsic nucleosome positioning properties of the central enhancer sequence assembled into mononucleosome core particles in vitro. We find that the enhancer DNA prefers three translational positions, each of which utilizes different rotational settings on the nucleosome core. We conclude that DNA binding factors that position nucleosomes may do so by stabilizing one configuration out of several that can be adopted by the underlying DNA, and that the potential exists for different positions to be stabilized at different stages of development.

An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array.

Nucleosomes positioned over promoters are usually inhibitory to protein binding and activity. We analyzed at the nucleotide level of resolution the nucleosomal organization of a distal, liver-specific enhancer in various mouse tissues and found that the enhancer exists in an array of three precisely positioned nucleosomes only in liver chromatin, where the enhancer is active. In vivo footprinting reveals that essential transcription factor-binding sites are occupied on apparent nucleosome surfaces, in one case leading to a perturbed nucleosomal structure. A similar nucleosomal array is generated with an in vitro chromatin assembly system in which nucleosome positioning is dependent upon binding to the enhancer of proteins related to hepatocyte nuclear factor 3. We suggest that certain transcription factors can organize nucleosomal structures that define an active enhancer element.

Nutrient-responsive promoter elements of the V4 gene of Dictyostelium discoideum.

Expression of the V4 gene of Dictyostelium discoideum is required for the transition from growth to development in response to an altered nutrient environment. In addition, the expression itself is sensitive to the types and amounts of nutrients supporting growth. We describe the structure of the two copies of the V4 gene and the relationship between these genes and the two V4 mRNA species produced during growth. In addition, three regions were identified within the upstream sequences of the V4b gene that are important for proper transcription. At least two of the regions can, independently of the others, confer deactivation of transcription upon initiation of development and thus serve as redundant regulatory sequences. However, the regions are differentially responsive to the types and amounts of nutrients present in the cell's environment and thus are distinct from one another.

V4, a gene required for the transition from growth to development in Dictyostelium discoideum.

The V4 gene of Dictyostelium discoideum is regulated in a nutrient-dependent manner and is deactivated immediately upon the onset of development. V4 is expressed only during growth, but its expression is not required for growth. We propose that the V4 gene product plays a role in the transition from growth to development. We have tested this hypothesis by antisense mutagenesis. Cells transformed with a V4 antisense construct contained no detectable endogenous V4 mRNA. These cells grew normally, but they failed to aggregate. Under conditions which normally promote development, V4 antisense transformants failed to deactivate vegetative-specific genes. These cells also were unable to induce the expression of the cAMP cell surface receptor, the cyclic nucleic phosphodiesterase, and contact sites A, all of which are normally induced under such conditions. Surprisingly, cells transformed with a V4 sense construct displayed a similar morphological and biochemical phenotype as the antisense cells, whereas cells transformed with the parental vector exhibited a normal biochemical and morphological phenotype. These results demonstrate that expression of the V4 gene during growth is required for the proper initiation of development.

Structure, expression, and regulation of members of the developmentally controlled V and H gene classes from Dictyostelium.

We have examined the expression and structure of vegetative specific genes belonging to the V and H gene classes. Both classes of genes are deactivated at the onset of development by a reduction in the rate of transcription. Thus, the genes must be reactivated when the terminally differentiated spores germinate and the resulting amebae return to the vegetative state. During germination, activation of expression of most members of the V gene class was found to parallel the emergence of amoebae from the spore coats. The activation of the V genes did not occur when protein synthesis was inhibited. The timing of activation of the H genes was more heterogeneous and did not parallel emergence. H gene activation occurred even when protein synthesis was inhibited. V4 was found to be the only vegetative specific gene that was responsive to the presence of bacteria. V4 expression was induced by 25-100 fold via transcriptional activation when bacteria were added to amebae growing axenically. Isolation and sequence analysis of the corresponding genomic clones revealed that two V genes, V18 and V1, encode ribosomal proteins. Promoter analysis has delineated the sequences necessary for expression and regulation for several of the V and H genes. In all cases, expression was determined by sequences within the first several hundred base pairs of the transcription start site. For V18 and V14, a positive constitutive element was identified in addition to the sequences involved in regulation. Finally, all of the characterizations and findings are discussed in terms of postulated models for V and H gene expression and regulation.

Deactivation of gene expression upon the onset of development in dictyostelium discoideum.

Several genes that are deactivated upon the initiation of development of Dictyostelium discoideum have been identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady-state levels of their corresponding mRNAs as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. However, for about 5% of the genes (H genes), cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the transcription rate; normal rates of transcription for the H genes were dependent upon continued protein synthesis during vegetative growth and during development. Thus, two general regulatory classes exist for deactivation of gene expression upon initiation of development, one dependent and one independent of protein synthesis. Models concerning the control of expression of these two classes of genes are discussed here. Analysis of expression of these genes in mutant strains that are aggregation-deficient has also been performed, and the results lead to subdivisions of the classes.

Characterization of genes which are deactivated upon the onset of development in Dictyostelium discoideum.

We have identified and begun characterizations of the differential expression of 15 genes whose corresponding mRNA levels decrease during the preaggregative period of the developmental program of Dictyostelium discoideum. Upon the onset of development, the mRNAs decrease from 5- to 1000-fold over the first 8 hr. The rates of loss of each mRNA were similar to one another but distinct, and the decreases were dependent on progress through the developmental program. One exception to this dependency was observed, and the decrease in this mRNA was dependent on the absolute time after initiation of development instead of progress through development. With two exceptions, the decreases in mRNA levels were dependent on developmental conditions and were not seen when cells were shaken in starvation buffer. When the polysomal distributions of each species were examined, three classes were found: most showed no significant shifts off of polysomes upon initiation of development, two were characterized by a 20% shift to nonpolysomal RNA fractions upon development, and two gave a 40-50% shift. Collectively, these characterizations reveal differences in behavior which suggest that deactivation of genes upon initiation of development in Dictyostelium involves more than one regulatory pathway.