Biomedical subjects
Publications and source records attributed to M F Cohen.
The flavonoid naringin was found to induce the expression of hrmA, a gene with a symbiotic phenotype in the cyanobacterium Nostoc punctiforme. A comparative analysis of several flavonoids revealed the 7-O-neohesperidoside, 4'-OH, and C-2-C-3 double bond in naringin as structural determinants of its hrmA-inducing activity.
Patterning along the left/right axes helps establish the orientation of visceral organ asymmetries, a process which is of fundamental importance to the viability of an organism. A linkage between left/right and axial patterning is indicated by the finding that a number of genes involved in left/right patterning also play a role in anteroposterior and dorsoventral patterning. We have recovered a spontaneous mouse mutation causing left/right patterning defects together with defects in anteroposterior and dorsoventral patterning. This mutation is recessive lethal and was named no turning (nt) because the mutant embryos fail to undergo embryonic turning. nt embryos exhibit cranial neural tube closure defects and malformed somites and are caudally truncated. Development of the heart arrests at the looped heart tube stage, with cardiovascular defects indicated by ballooning of the pericardial sac and the pooling of blood in various regions of the embryo. Interestingly, in nt embryos, the direction of heart looping was randomized. Nodal and lefty, two genes that are normally expressed only in the left lateral plate mesoderm, show expression in the right and left lateral plate mesoderm. Lefty, which is normally also expressed in the floorplate, is not found in the prospective floor plate of nt embryos. This suggests the possibility of notochordal defects. This was confirmed by histological analysis and the examination of sonic hedgehog, Brachyury, and HNF-3 beta gene expression. These studies showed that the notochord is present in the early nt embryo, but degenerates as development progresses. Overall, these findings support the hypothesis that the notochord plays an active role in left/right patterning. Our results suggest that nt may participate in this process by modulating the notochordal expression of HNF-3 beta.
Although gap junctions are not known to be important in mediating cell-cell interactions amongst migratory cells, our studies showed that the connexin 43 (Cx43) gap junction gene is widely expressed in mouse neural crest cell lineages. Using in situ hybridization analysis, Cx43 expression was detected in presumptive neural crest cells emerging from the neural folds of the early postimplantation embryo. Neural crest expression of the Cx43 gap junction gene was also indicated by the analysis of transgenic mice containing a lacZ reporter construct driven by the Cx43 promoter. In neural tube explant cultures of these transgenic mice, lacZ expression was observed in the emerging neural crest outgrowths. Whole mount X-gal staining of these transgenic embryos at various stages of development showed lacZ expression in neural crest cells distributed along the entire craniocaudal axis, with expression found in both cranial and trunk neural crest cells contributing to a wide range of embryonic tissues. This included presumptive cardiac neural crest cells localized in the heart. In light of the widespread expression of Cx43 in neural crest cell lineages, dye injection studies, were carried out to determine if neural crest cells are functionally coupled via gap junctions. Such studies revealed extensive dye coupling among presumptive neural crest cells, thus demonstrating that these migratory cells are indeed gap junctional communication competent. In total, these observations suggest that gap junctions may play a role in mouse neural crest development. This possibility is particularly intriguing given the recent finding that the Cx43 knockout mice die of defects associated with the outflow tract [Reaume et al., 1995], a region of the heart in which neural crest cells are required for normal development.
The Gja1 gene encoding the gap junction connexin 43 (Cx43) is dynamically regulated during limb morphogenesis. Transcript expression is found in many regions of the limb bud known to be important in regulating limb growth and patterning. In the newly emerged limb bud, Gja1 transcripts are first expressed in the ventrodistal margin of the ectoderm, and later transcript expression is localized to the apical ectodermal ridge (AER). Interestingly, transcript expression in the ventrodistal ectoderm is initiated left/right asymmetrically, with some strain backgrounds showing reverse sidedness in the fore vs. hindlimb buds. In legless, a mouse mutant exhibiting both limb and left/right patterning defects, Gja1 transcripts could not be detected in this region. However, in the i.v./i.v. embryo, a mutant with randomization of body situs the same pattern of Gja1 asymmetry was found in the limb ectoderm regardless of body situs. This suggests that Gja1 transcript expression is not directly linked to signaling pathways involved in specification of the left/right axis. In addition to transcript expression in the apical ectodermal ridge, Gja1 transcripts were also found at high levels in the ventral ectoderm. In the limb bud mesenchyme, Gja1 transcripts were distributed in a posterior distal gradient, coincident with tissue known to have polarizing activity. With limb outgrowth and the initiation of limb mesenchyme condensation. Gja1 transcripts were localized in the presumptive progress zone, and in the condensing mesenchyme. In more proximal regions of the limb where mesenchyme differentiation has been initiated, Gja1 transcripts were expressed only in the outer mesenchymal cells comprising the presumptive perichondrium. Further analysis of transgenic mice ectopically expressing Wnt-1 in the limb mesenchyme revealed alterations in the pattern of Gja1 transcript expression in conjunction with the perturbation of limb mesenchyme condensation and differentiation. Together, these findings indicate that Cx43 gap junctions may mediate cell-cell interactions important in cell signaling processes involved in limb growth and patterning.
A Tn5-1063-derived mutant of Nostoc punctiforme strain ATCC 29133 was unable to fix N2 in air although it reduced acetylene in the absence of O2. Mutant strain UCD 307 formed cells morphologically similar to heterocysts, but it failed to synthesize the characteristic heterocyst glycolipids. Sequence analysis around the site of insertion revealed an ORF of 3,159 base pairs, termed hglE. hglE putatively encodes a 115.4-kDa protein containing two domains with conserved amino acid sequences identified with acyl transferase and the chain length factor variation of beta-ketoacyl synthase active sites. These active sites are characteristic of polyketide and fatty acid synthases. The N. punctiforme strain 29133 hglE gene is transcribed only under nitrogen-limiting growth conditions. The hglE gene, or similar sequences, was found in all other heterocyst-forming cyanobacteria surveyed and was absent in unicellular Synechococcus sp. strain PCC 7942. Based on these results, we propose that the synthesis of heterocyst glycolipids follows a pathway characteristic of polyketide synthesis and involves similar large, multienzyme complexes.
Transposon-generated mutant strain UCD 328 of Nostoc punctiforme strain ATCC 29133 has a phenotype of an increased sensitivity to a hormogonium-inducing factor exuded by a symbiotic plant partner, Anthoceros punctatus, and an initial increased hormogonium-dependent infection of the plant. Sequence analysis showed that the transposition site in strain UCD 328 lies within a 1,251-bp open reading frame (ORF), designated hrmA, that displays no significant similarity to known database sequences. A second, 837-bp ORF (hrmU) ends 2 bp 5' from the start of hrmA and has the signature sequences belonging to a family of NAD(P)H-dependent oxidoreductases. Strains having insertional mutations in hrmU or hrmA reproduce the strain UCD 328 phenotype. Transcriptional fusions of luxAB to hrmU or hrmA show an 8- to 10-fold peak increase in luciferase activity 13 to 20 h after the start of incubation in the presence of an aqueous extract of A. punctatus. A promoter induced by the extract was deduced to be between 2.0 to 3.4 kb from the translational start of hrmU. A multicopy plasmid that contains hrmUA within a 6.2-kb fragment conferred an increased infection phenotype on wild-type N. punctiforme 29133. This plasmid and another plasmid containing 4.4 kb of DNA 5' of the transposition site prevented extract-dependent induction of hrmA-luxAB transcription in strain UCD 328, implicating titration of some trans-activator(s) by the cloned fragments. We hypothesize a role for hrmUA in the inhibition of hormogonium formation by the metabolism of an unknown hormogonium-regulating metabolite.
Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25-31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194-2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 transgenic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.
Strain UCD 311 is a transposon-induced mutant of Nostoc sp. strain ATC C 29133 that is unable to fix nitrogen in air but does so under anoxic conditions and is able to establish a functional symbiotic association with the hornwort Anthoceros punctatus. These properties of strain UCD 311 are consistent with previous observations that protection against oxygen inactivation of nitrogenase is physiologically provided within A. punctatus tissue. Upon deprivation of combined nitrogen, strain UCD 311 clearly differentiates heterocysts and contains typical heterocyst-specific glycolipids; it also makes apparently normal akinetes upon phosphate starvation. Sequence analysis adjacent to the point of the transposon insertion revealed an open reading frame designated devR. Southern analysis established that similar sequences are present in other heterocyst-forming cyanobacteria. devR putatively encodes a protein of 135 amino acids with high similarity to the receiver domains of response regulator proteins characteristics of two-component regulatory systems. On the basis of its size and the absence of other functional domains, DevR is most similar to CheY and Spo0F. Reconstruction of the mutation with an interposon vector confirmed that the transposition event was responsible for the mutant phenotype. The presence of wild-type devR on a plasmid in strain UCD 311 restored the ability to fix nitrogen in air. While devR was not essential for differentiation of akinetes, its presence in trans in Nostoc sp. strain ATCC 29133 stimulated their formation to above normal levels in aging medium. On the basis of RNA analysis, devR is constitutively expressed with respect to the nitrogen source for growth. The devR gene product is essential to the development of mature heterocysts and may be involved in a sensory pathway that is not directly responsive to cellular nitrogen status.
Nostoc sp. strain ATCC 29133 (PCC 73102; Nostoc 29133) is a symbiotically-competent, facultatively heterotrophic, diazotrophic cyanobacterium with the capacity to differentiate specialized cells such as heterocysts, akinetes and hormogonial filaments. We have optimized several methods for physiological and molecular genetic analysis of Nostoc 29133. By use of a Tn5 derivative, Tn5-1063 (Km(r)Bm(r)Sm(r)), delivered by conjugation from Escherichia coli, antibiotic-resistant mutants of Nostoc 29133 were generated at a frequency of approximately 1 x 10(-6), 0.4% of which expressed a nitrogen fixation (heterocyst) defective phenotype. Mutant strain UCD 328 was isolated after co-culture of 86 Nostoc 29133::Tn5-1063 clones with the symbiotic plant partner, Anthoceros punctatus; strain UCD 328 expressed a symbiotic phenotype of increased frequency of hormogonia-dependent infection. The transposon and flanking genomic DNA was recovered from strain UCD 328, the mutation and phenotype reconstructed by homologous recombination in Nostoc 29133, and the transposition site identified from a Nostoc 29133 genomic library. Transposon mutagenesis has thus provided the means for isolation and identification of developmental and symbiotic-specific genes of Nostoc 29133.
Experiments were performed to determine the effect of coherent dynamic frequency change among stimulus components on detection of one of those components. Detectability of a frequency glide signal, centered at 660 Hz, was measured in the presence of two additional frequency glides centered at 220 and 440 Hz. Results show that the signal is most difficult to detect when it is changing coherently with the other stimulus components, and that detectability gradually improves as the frequency change among stimulus components becomes less coherent. A control experiment indicated that detectability of the signal is influenced somewhat by the average spectral distance between the signal and the other stimulus components. In order to separate the effects of dynamic frequency change from those of altering harmonic relationships, the experiment was repeated with stimulus components not harmonically related. Results are similar in pattern, though with somewhat smaller shifts. A final experiment was performed to determine if the observed shifts in detectability might be due to changes in the maximum spectral distance between signal and masker when their frequencies are changing incoherently. Results of this experiment indicate that detectability improves when frequency change of signal and masker is not coherent, even when maximum spectral distance does not change. These data indicate the likelihood that the auditory system is able to use coherent dynamic frequency change among stimulus components.
An experiment was performed to determine if the mechanism that mediates comodulation masking release (CMR) is associated with that used to improve detection by the masking-level difference (MLD). The experiment consisted of first improving detectability of a masked diotic tone burst by adding a synchronous noise band at another frequency region (CMR), and then measuring an MLD in the usual manner, by inverting the tone-burst signal to one ear. Results indicate that a substantial MLD can be measured for a signal whose detectability has already been improved by CMR. However, that MLD (9 dB) is smaller than that measured in random noise (14 dB). Put another way, a small CMR (4 dB) can be produced even when the detectability of a stimulus has already been improved due to the MLD. These data are in general agreement with those of Hall et al. [J. Acoust. Soc. Am. 83, 1839-1845 (1988)] and Schooneveldt and Moore [J. Acoust. Soc. Am. 85, 262-272 (1989)].
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Seeking to resolve conflicting literature on cytoskeletal structure in mammalian "primitive" generation erythrocytes, we have utilized the circulating blood of developing marsupials. In young of the Tammar Wallaby (Macropus eugenii) and the Gray Short-tailed Opossum (Monodelphis domestica), relatively large, nucleated primitive erythrocytes constituted nearly 100% of the circulating population at birth (= day 0) and in fetuses (Tammar) several days before birth. These cells were discoidal or elliptical, and flattened except for a nuclear bulge. Their cytoskeletal system, consisting of a marginal band of microtubules enclosed within a cell surface-associated network (membrane skeleton), closely resembled that of non-mammalian vertebrate erythrocytes. By day 2 or 3, much smaller anucleate erythrocytes of "definitive" morphology, lacking marginal bands, appeared in abundance. These accounted for greater than 90% of the circulating population of both species by day 6-8. Non-nucleated erythrocytes of a different type, constituting 1-6% of the cells in most blood samples up to day 7, were identified as anucleate primitives on the basis of size, shape, and presence of a marginal band. Thus, loss of erythrocyte nuclei in mammals appears to begin earlier than generally recognized, i.e., in the primitive generation. Counts of these anucleate primitives in young of various ages implicated nucleated primitives as their probable source. Pointed erythrocytes, occasionally found in younger neonates of both species, occurred in greatest number in fetuses (Tammar) prior to birth. This is in accord with previous work on non-mammalian vertebrates suggesting that such cells are morphogenetic intermediates. The results confirm the long-suspected similarity between mammalian primitive erythrocytes and the nucleated erythrocytes of all non-mammalian vertebrates.
An experiment was performed to study the interaction of two narrow-band noises having correlated temporal envelopes. The detection threshold of a 100-Hz-wide noise-band signal was measured at different center frequencies in the presence of a continuous 100-Hz-wide noise band having a center frequency of 1000 Hz. The two noise bands had either correlated or independent temporal envelopes. Measured signal detection thresholds are lower when the two noise bands are independent, but the magnitude of this difference is not a simple function of the frequency separation between the two noise bands.
This study was designed to investigate the effects of masker level and frequency on binaural detection and interaural time discrimination. Detection and interaural time discrimination of a 700-Hz sinusoidal signal were measured as a function of the center frequency and level of a narrow-band masking noise. The masker was a continuous, diotic, 80-Hz-wide noise that varied in center frequency from 250 to 1370 Hz. In the detection experiment, the signal was presented either diotically (NoSo) or interaurally phase reversed (NoS pi). In the interaural time discrimination experiment, the signal level needed to discriminate a 30-microseconds interaural delay was measured. As would be expected, the presence of the masker has a greater effect on NoSo detection than NoS pi detection, and for masker frequencies at or near the signal frequency. In contrast, interaural time discrimination can be improved by the presence of a low-level masker. Also, performance improves more rapidly as the signal/masker frequency separation increases for NoSo detection than for interaural time discrimination and NoS pi detection. For all three tasks, significant upward spread of masking occurs only at the highest masker level; at low masker levels, there is a tendency toward downward spread of masking.
A series of experiments was performed to study the ability of the ear to code the temporal envelope of a waveform as demonstrated by comodulation masking release (CMR). The stimulus for all experiments was composed of a tone-burst signal, a 100-Hz-wide masker band centered at the signal frequency, and a second 100-Hz-wide noise band of variable frequency, the cue band. The cue band had a temporal envelope which was either correlated with or independent of that of the masker. The signal was a 100-Hz tone burst for most experiments. For the monotic stimulus, the correlated cue band results in lowered signal detection thresholds over a range extending from around 2/3 oct below the signal frequency to 1/3 oct above that frequency. When measured dichotically, with the signal and masker band in one ear and the cue band in the opposite ear, that effective range is expanded but the detection threshold shifts are a bit smaller. The greatest CMR is observed when the stimulus is presented diotically. With regard to effects of level and frequency, our data show CMR increasing with increasing stimulus level for a cue band lower in frequency than the signal, but show little effect of level for a cue band higher in frequency. Similarly, CMR increases with increasing stimulus frequency when the cue band is lower in frequency, but shows little effect of frequency for a cue band higher in frequency.
A series of experiments was performed to examine the extent to which precision of interaural time discrimination depends on the sound-pressure level (SPL) and/or sensation level (SL) of the signal. All experiments used a tone burst signal and a continuous white noise masker, which was either diotic or interaurally phase reversed. Results of the first experiment indicate that (1) at equal signal SLs, interaural time and intensity discrimination is more precise when measured with the added diotic noise, and (2) addition of the phase reversed noise, previously shown to cause less precise interaural time discrimination, has a similar effect on interaural intensity discrimination. In the second experiment, interaural time JNDs for a signal of constant SPL were measured as a function of noise level. Results show that a low-level diotic noise can benefit interaural time discrimination, particularly at 500 Hz. The third and fourth experiments were performed to measure interaural time discrimination as a function of increasing signal SPL but constant signal-to-noise ratio. The data show the JND decreasing with increasing signal SPL at nearly the same rate with or without the added noise, indicating that an increase in signal-to-noise ratio is not necessary for improved discrimination.