Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. 1,6-Diphenyl-1,3,5-hexatriene in lipid bilayers

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L., & Brand, L. (1986) Biochemistry (preceding paper in this issue)] for studying rotational microheterogeneity of probe location in lipid bilayer systems is described. Anisotropy decay associated spectra are derived from experimentally obtained polarized emission components. "Early" difference spectra (IV - IH) contain contributions from both fast and slow rotors, while "late" difference spectra predominantly reflect the emission from slowly rotating fluorophores. Anisotropy decay associated spectra have been used to resolve the emission spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) imbedded within a known rotationally heterogeneous mixture of two vesicle types (L-alpha-dimyristoyllecithin and L-alpha-dipalmitoyllecithin). At 29 degrees C, diphenylhexatriene within pure dimyristoyllecithin vesicles rotates rapidly, with a small r infinity, while diphenylhexatriene in dipalmitoyllecithin vesicles exhibits a large r infinity. Spectra for diphenylhexatriene imbedded in the two vesicle types show small but significant spectral differences. A spectrum of a mixture of the two vesicle types with DPH lies between these characteristic component spectra. The spectrum extracted for "immobilized" probes in the mixture correctly overlays the dipalmitoyllecithin spectrum. Further studies have shown that diphenylhexatriene exhibits more than one emission anisotropy decay associated spectrum in vesicles of a single lipid type, when that lipid is near its phase transition temperature. Diphenylhexatriene apparently inhabits more than one rotational environment even in these "homogeneous" vesicle preparations.     

Reflex control of inspiratory duration in newborn infants

We applied graded resistive and elastic loads and total airway occlusions to single inspirations in six full-term healthy infants on days 2-3 of life to investigate the effect on neural and mechanical inspiratory duration (TI). The infants breathed through a face mask and pneumotachograph, and flow, volume, airway pressure, and diaphragm electromyogram (EMG) were recorded. Loads were applied to the inspiratory outlet of a two-way respiratory valve using a manifold system. Application of all loads resulted in inspired volumes decreased from control (P less than 0.001), and changes were progressive with increasing loads. TI measured from the pattern of the diaphragm EMG (TIEMG) was prolonged from control by application of all elastic and resistive loads and by total airway occlusions, resulting in a single curvilinear relationship between inspired volume and TIEMG that was independent of inspired volume trajectory. In contrast, when TI was measured from the pattern of airflow, the effect of loading on the mechanical time constant of the respiratory system resulted in different inspired volume-TI relationships for elastic and resistive loads. Mechanical and neural inspired volume and duration of the following unloaded inspiration were unchanged from control values. These findings indicate that neural inspiratory timing in infants depends on magnitude of phasic volume change during inspiration. They are consistent with the hypothesis that termination of inspiration is accomplished by an "off-switch" mechanism and that inspired volume determines the level of vagally mediated inspiratory inhibition to trigger this mechanism.     

Respiratory-related cortical potentials evoked by inspiratory occlusion in humans

It has long been recognized that humans can perceive respiratory loads. There have been several studies on the detection and psychophysical quantification of mechanical load perception. This investigation was designed to record cortical sensory neurogenic activity related to inspiratory mechanical loading in humans. Inspiration was periodically occluded in human subjects while the electroencephalographic (EEG) activity in the somatosensory region of the cerebral cortex was recorded. The onset of inspiratory mouth pressure (Pm) was used to initiate signal averaging of the EEG signals. Cortical evoked potentials elicited by inspiratory occlusions were observed when C3 and C alpha were referenced to CZ. This evoked potential was not observed with the control (unoccluded) breaths. There was considerable subject variability in the peak latencies that was related to the differences in the inspiratory drive, as measured by occlusion pressure (P0.1). The results of this study demonstrate that neurogenic activity can be recorded in the somatosensory region of the cortex that is related to inspiratory occlusions. The peak latencies are longer than analogous somatosensory evoked potentials elicited by stimulation of the hand and foot. It is hypothesized that a portion of this latency difference is related to the time required for the subject to generate sufficient inspiratory force to activate the afferents mediating the cortical response.     

Effects of maternal administration of diethylstilbestrol and estradiol on the newborn guinea pig

The subcutaneous injection of diethylstilbestrol (2.5 micrograms/day) into pregnant guinea pigs from the 28th or 40th day to term resulted in an accelerated tempo of differentiation of the genital tract of the female offspring at birth, as well as intense estrogenic stimulation. Estradiol (50 micrograms per day) injected over the same period caused insignificant estrogenic stimulation in the newborn. The normal embryogenesis of the genital tract is described and a pattern of caudocranial differentiation identified. The normal genital tract is described as a basis for the analysis of results in the newborn. Comparative studies in other animals are discussed.     

Nucleolar necklace formation in response to hemolymph ecdysteroid peaks.

Previous work on the last (fifth) larval stadium of Calpodes showed two phases of elaboration of epidermal nucleoli correlated with RNA synthesis, the first after ecdysis at the beginning of the intermolt and the second near the end of the stadium prior to molting. Both phases followed periods of elevated hemolymph ecdysteroid. The demonstration of four hemolymph ecdysteroid peaks and an improvement in the bismuth-staining procedure for nucleoli has prompted further study of nucleolar changes in relation to hemolymph edcysteroids. We have found that three of the four ecdysteroid peaks (I, II and IV) are followed by nucleolar changes. The exception is the commitment peak (III) for which there is no corresponding nucleolar change. The three nucleolar cycles are similar in their essential features. An intercycle nucleolus consists of one or a few irregularly shaped particles that become more densely stained and condense into a knot at the beginning of each cycle. The knot unfolds into a necklace which beomes beaded as it elongates to a length of about 23 mum. Cells have one or two, rarely more, necklaces presumably depending on their ploidy. At the end of the cycle the necklaces contract, becoming coarser and fragmented before they condense to the intercycle condition of central irregular cores. Whereas nucleolar necklaces are a general response to hemolymph ecdysteroids, mitoses are locally determined and are imposed over other nuclear activities at any time in the third nucleolar cycle.     

The structure of epidermal feet during their development

Epidermal cells in Calpodes and other insects form basal processes or feet that at first extend axially and later shorten at the same time as the larval segment shortens to the pupal shape. The feet grow into spaces at the surfaces of other cells to make a basal interlacing meshwork of cellular extensions that are combined mechanically by their desmosomal attachments to cell bodies above and to the basal lamina below. Microtubules and microfilaments are linked to these junctions by a reticular fibrous matrix. Gap junctions on the feet may couple cells that are several cell bodies removed from one another. The meshwork is also a sieve separating the hemolymph from the spaces between cells to form an intercellular compartment. Entry to the intercellular compartment is through the sieve made by the negatively charged basolateral cell surfaces that can prevent the entry of positively charged molecules such as cationic ferritin. As the cells become columnar, coincident with the metamorphic change in segment shape, the feet shorten and pack more densely together. At this time the basal lamina buckles axially as if responding to contraction of the feet. Segment shape change involves cell rearrangement and relative cell movement, necessitating the transient loss of plasma membrane plaque attachments to the cuticle apically and the loss of junctions laterally. Gap junctions involute in characteristic vacuoles. The metamorphic reduction in cell surface area coincides with the loss of basolateral membrane in smooth tubes and vesicles and the turnover of the apical surface in multivesicular bodies. New apical plasma membrane plaques and new lateral and basal junctions stabilize the cells in their pupal positions.