Mixed (chemical and electrical) synapses on frog spinal motoneurons. Freeze-fracture replicas and ultrathin sections were used to characterize the gap junctions on the somata and large dendrites of frog motoneurons found earlier by Sonnhof et al. (1977). In freeze-fracture replicas one of the specific features of these relatively frequent gap junctions is the presence of circular regions of non-junctional membrane ("fenestrae") within areas of typical gap junction appearance displaying P-face particles or E-face pits. Such "fenestrated" gap junctions are mostly associated with membrane specializations indicative of the active zone of a chemical synapse (including vesicle attachment sites in non anaesthetized animals) to constitute mixed synapses. These findings could be verified in ultrathin sections, which revealed that the vesicles of the chemical component of the mixed synapses were spherical and agranular. Our results suggest that the mixed synapses are predominantly axo-somatic and axo-dendritic. The existence of dendro-dendritic gap junctions in the ventral horn region as described by Sotelo and Taxi (1970) was verified in ultrathin sections; they were rare, solely electrotonic in character, and probably represent the morphological basis for the VR-EPSP (Katz and Miledi, 1963; Kubota and Brookhart, 1963), i.e. electrotonic coupling between motoneurons of different spinal segments (Washizu, 1960). Electrotonic coupling can also be demonstrated between motoneurons and afferent fibers of the dorsal root and the lateral column. Electrotonic potentials recorded within motoneurons during electrical stimulation of dorsal root or lateral column precede the chemical postsynaptic potentials; after Mn2+-blockade of chemical synaptic transmission, the electrotonic component persists. Some fibers of these afferent pathways are therefore assumed to act monosynaptic on the motoneuron via mixed axo-somatic and axo-dendritic synapses.


The oligodendrocytic junctional complex. The junctional complex of oligodendrocytes was studied by means of different electron microscopical techniques. This complex is composed of the following junctional membrane formations: 1) tight junctional domains in the oligodendrocytic membrane near the some of the cells, 2) fasciae occludentes or focal tight junctions on the outer oligodendrocytic loop of myelin and on the outermost myelin membrane, 3) gap junctions of considerable size variations, either on membranes near the soma or on peripheral oligodendrocytic processes, and 4) non-paranodal transverse bands. The different types of oligodendrocytic junctions are discussed in terms of their functional implications.


Ultrastructural radioautography of the incorporation of tritiated leucine by the rat adrenal medulla in vivo. Sections of tissues from the adrenal medullae of young rats were subjected to radioautography after a single intravenous injection of L-leucine 4, 5(3)H to identify the sites of synthesis and follow the migration of newly-formed proteins in both adrenaline-storing (A) and noradrenaline-storing (N) cells. As early as 2 min after injection of leucine 3H, the label was highest in the rough endoplasmic reticulum (RER) of A and N cells, suggesting that cisternal ribosomes are sites of protein synthesis. By 5 and 10 min, much of the label had migrated from the RER into the Golgi complex of both cell types. Some label was already present over the secretory granule matrix (chromogranins) by 2 min but the peak was reached at 1 h in both A and N cells. By 4 h, the label over the secretory granules had diminished, indicating a release of newly-synthetized chromogranins outside the cells. The label over the hyaloplasm was relatively high at 2 min but it decreased rapidly to low levels. In contrast, the label over the cell surface continually increased to reach the highest levels among all organelles at 4 h in both cell types. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the hyaloplasm, before reaching the surface of A and N cells.


Quantitative analysis of epithelial changes during wound healing in palatal mucosa of guinea pigs. The epithelium of wounded guinea pig palate was subjected to stereologic analysis. A total of 18 biopsies (animals) were used. Biopsies were taken at 18, 48, 96 and 120 h after wounding. Point counting procedures were employed to analyse electron micrographs sampled from one (18 h) or two epithelial strata (48, 96 and 120 h). The essential modulations in epithelial structure as wound healing proceeds were as follows: During the early phases characterized by formation and advancement of epithelial lips (18 and 48 h), migrating cells converged towards a cell type which structurally was less differentiated than normal basal cells. This alteration was expressed by a decrease in volume density of cytoplasmic organelles, mainly mitochondria, free ribosomes and tonofilament bundles, coupled with an increase in volume density of lysosomal bodies. Concomitantly, the volume density of cytoplasmic ground substance rose markedly. Subequent to fusion of contralateral migratory lips (96 and 120 h) reversion to normal epithelial structure was indicated by the increment in magnitude of basal cell parameters. Further structural density gradients from basal towards upper cell layers appeared. This pattern was mainly displayed by mitochondria, free ribosomes, and tonofilament bundles. The magnitude and gradation of most tissue and cell parameters were not yet re-established at 120 h. The density of tonofilament bundles and the density level of cytoplasmic ground substance in particular deviated.


Cell proliferation in skeletal muscle following denervation or tenotomy. A series of autoradiographic studies. Autoradiographic experiments using 3H-thymidine were designed to analyse cell proliferation which occurs in skeletal muscle after denervation and after tenotomy. In mouse tibialis anterior and tongue muscles during the first 24 h after denervation or tenotomy labelling levels were low and did not differ significantly from sham operated control muscles. By 48 h after denervation and tenotomy of tibialis anterior muscles, increased levels of labelling occurred in both muscle and connective tissue nuclei. Daily pulse labelling for 7 days after denervation produced a labelling level which was 8 times that of sham operated controls, 25--30% of the total nuclear population being labelled. Denervated muscles had twice the level of labelling compared to tenotomised muscles. These results provide conclusive evidence that both denervation and tenotomy stimulate cell proliferation in skeletal muscle and it is suggested that the increased numbers of labelled muscle nuclei are likely to be the result of mitotic activity in muscle satellite cells.


High resolution radioautographic study of newly formed protein in striated muscle with emphasis on red and white fibres. The elaboration and distribution of newly formed proteins in the striated muscle of 21-day-old mice were investigated by quantitative radioautography at intervals between 2 and 240 min after intravenous injection of tritiated leucine. In radioautographs, the localization and the relative label concentration were comparatively estimated for the different components of mitochondria-rich fibres, in particular of red fibres, from the tibialis anterior muscle and of mitochondria-poor fibres from the oesophageal muscle. As early as 2 min after injection, radioactivity was detected over the nucleus, the polysome-rich sarcoplasm, the A and I bands, the Z lines, and the mitochondria in the two fibre types. Label localization did not change with time. The relative label concentration increased similarly in the polysome-rich sarcoplasm and the A and I bands of both fibre types within 30 min after injection, a confirmation that biosynthesis of myofibrillar proteins takes place rapidly. In each case, concentration was higher in the Z lines than in the I bands, and higher in the I bands than in the A bands, thus showing "in vivo" that the rates of synthesis of sarcomere protein components are not uniform. However, the relative label concentration was found to be higher in the Z lines of mitochondria-poor fibres than of mitochondria-rich fibres: this suggests that a higher synthetic rate of Z line protein, and probably of alpha actinin, is characteristic of the first type. Inversely, the concentration was higher in the mitochondria of mitochondria-rich fibres. This lead to the belief that high rate of protein synthesis in these organelles may account for the high rate of label incorporation into this type of fibre.


Degradation of the radula in the snails Biomphalaria glabrata Say and Limnaea stagnalis L. (Gastropoda, Pulmonata). The radula of snails is formed at the posterior end of the radular gland or pocket, and degraded at the same rate at its anterior end. Degradation is due to different secretory activities of the inferior epithelium of the radular gland. Its secretions seem to degrade enzymatically the matrix of the radular membrane and basal plates of teeth, leaving only chitin containing microfibres and degradation products. The sclerotized parts of the teeth remain unchanged, but as they are now only loosely connected with the radular membrane. they are torn off easily during feeding movements. The rest of the degraded and frayed radular membrane and the subradular membrane are also lost by abrasion during feeding. The cells of the inferior epithelium are connected with each other by septate desmosomes and an elaborate system of deep lateral interdigitation which may provide tensil strength. Extrusion of degraded cells of the inferior epithelium into the subradular membrane takes place, although the thick basal lamina forms a continuous sheath which is closely adjoined to the basal parts of the inferior epithelium. Nerve fibres containing vesicles with electron dense neurosecretory material (deduced from the diameter of 200--250 nm) are attached to this sheath or penetrate into it; they may be involved in the regulation of production and degradation processes during radula replacement. Problems of the forward transport of radula and inferior epithelium are discussed.


The pineal gland of the gerbil, Meriones unguiculatus. I. An ultrastructural study. Electron microscopy was employed in a study of the pineal gland of the Mongolian gerbil (Meiones unguiculatus). It was determined that the gerbil pineal gland contains pinealocytes and glial cells with the pinealocytes being the predominant cell type. The pinealocytes contain numerous organelles traditionally considered as being either synthetic or secretory in function such as an extensive Golgi region, smooth (SER) and rough (RER) endoplasmic reticulum, secretory vesicles and microtubules. Other cytoplasmic components are also present in the pinealocytes (synaptic ribbons, subsurface cisternae) for which no function has been assigned. Dense-cored vesicles are rare. Vacuolated pinealocytes are present and appear to be intimately associated with the formation of the pineal concertions. Evidence presented supports the proposal that the concretions form within the vacuoles. Once the concretions reach an enlarged state, the vacuolated pinealocytes break down and the concretions are thus extruded into the extracellular space where they apparently continue to increase in size. The morphology of the glial cells was interpreted as indicative of a high synthetic activity. The glial cells contain predominantly the rough variety of endoplasmic reticulum and form an expansion around the wide perivascular area.


The neuronal connections of the frontal ganglion of the cockroach Periplaneta americana. A histological and iontophoretical study. The frontal ganglion of the cockroach Periplaneta americana was studied histologically and its neuronal pathways were mapped by use of axonal cobalt iontophoresis. Neurons and fiber tracts of the frontal ganglion are directly linked with different regions of the central nervous system (tritocerebrum, protocerebrum, subesophageal ganglion) and with the more caudal parts of the stomatogastric nervous system (hypocerebral ganglion, nervus oesophagei).


Effects of reserpine administration on the fine structure of the rat pars intermedia. Hormone release from the pars intermedia is under inhibitory control of the hypothalamus. Control may be mediated via dopaminergic fibers which directly contact secretory cells. Administration of reserpine in vivo to adult male rats for four consecutive days results in major alternations in pars intermedia cytology. Cells show expanded areas and whorls of rough endoplasmic reticulum, as well as extensive Golgi zones with numerous dense granules. Some nerve fibers exhibit alterations in vesicle content, while others retain a more normal appearance. Freeze-fracture of glands from reserpine-treated animals provides evidence for exocytosis of granules, although such phenomena are not observed in thin sections. The ultrastructural findings suggest that reserpine alters the content of local inhibitory neurotransmitters in the pars intermedia, leading to unrestrained hormone release, followed by renewed granule synthesis.


Sarcotubular development in dystrophic skeletal muscle cells. Dilations of the sarcotubular system and misaligned myofilaments have been reported as early indicators of muscular dystrophy in skeletal muscle. Since the developing tubular component is believed instrumental in initial myofilament alignment during myogenesis, tubular development is evaluated using normal and dystrophic chick embryo skeletal muscle and cultures of normal and dystrophic embryonic pectoral muscle incubated in the presence of horse spleen ferritin. Comparisons of the findings show that periodic tubules are absent from dystrophic somitic muscle and that invaginating tubules from the sarcolemma are found in fewer, randomly located areas of dystrophic pectoral muscle cells. The results indicate that the tubular component is not involved in the bizarre vesiculations seen in mature dystrophic muscle, however, the malalignment of dystrophic myofilaments is probably the result of the poorer development of the T system in this muscle.


The antral gastrin-producing G-cell: biochemical and ultrastructural responses to feeding. The effect of feeding on serum and antral immunoreactive gastrin (IRG) concentrations and on the ultrastructural appearance of antral G-cell granules has been examined. Serum and tissue IRG concentrations were dependent upon the length of time (12 or 48 h) the rats had been fasted before receiving food; IRG release was biphasic; the first peak was more pronounced in rats fasted 12 h. Antral tissue IRG content increased significantly postprandially. An initial depletion of antral IRG was seen in rats fasted 48 h. Examination of the subcellular distribution of antral IRG revealed more of the 5-15 min postprandal total IRG in the cytoplasm and less in the secretory granules. Ultrastructurally, G-cells from fasting rats contained mainly electron-dense granules. Five minutes postprandially numerous electron-lucent granules were observed. More electron dense granules were apparent 60 and 120 min postprandially. Fasting rats had the highest G-cell granule density index; a significantly lower index was observed 5 min postprandially. Indices at 60 and 120 min postprandially increased but were still lower than the fasting index. These studies indicate that gastrin biosynthesis is necessary for food stimulated gastrin release and that the electron density of the G-cells granules is not an accurate reflection of the G-cell gastrin content.


The myotendinous junction of the smooth feather muscles (mm. pennati). A light and electron microscopic study on a myoelastic system. Smooth feather muscles (mm. pennati) consist of bundles of smooth muscle cells which are attached to the feather follicles by short elastic tendons. In addition, some muscle bundles are interrupted by elastic tendons. The elastic tendon is composed of longitudinally arranged elastic fibers which branch and wavy bundles of collagen fibrils. Smooth muscle cells of the muscle bundles are attached to each other by desmosome-like junctions and by fusion of the basal laminae. The cytoplasm of the muscle cells is characterized by conspicuous thick filaments and abundant thin and intermediate filaments. These are attached to band-like dense patches (dense bands) at the plasma membrane which are particularly broad at the tapering end of the muscle cell. The contact surface between smooth muscle cells and their elastic tendon is considerably increased (i) by deep finger-like invaginations and indentations located at the tapering muscle end, and (ii) by branching of the coarse elastic fibers into slender processes, which are attached to the richly folded surface of the muscle cell endings by peripheral microfibrils. This intimate interlocking closely resembles the myotendinous junctions in skeletal muscle. In addition to fibroblasts and fibrocytes, the myotendinous junction of the young growing chicks contains numerous so-called myofibroblasts, which are suggested to represent smooth muscle cells differentiating into fibroblasts of the developing tendon.


The adaptation of a two-compartment cell renewal system to external demands. A study of the rat incisor inner enamel epithelium. The inner enamel epithelium (IEE) covers the labial tooth aspect as a one cell layer which, when cut sagittally, appears as a longitudinal cell column extending from the tooth origin toward the periphery. Following sudden tooth shortening, the IEE responds by an increased cell production which later declines below normal values. The perturbation affects all cell kinetic parameters; the progenitor compartment, which initially increases, diminishes in size toward end of the experiment. The cell cycle transition times, which initially decline, rise toward the end of the experiment. The mean normal daily cell production rate of 70 cell % (i.e. 70 cells are produced by 100 progenitors) increases to 111 cell % and then declines to a low of 51 cell %. The IEE response typifies the behavior of other cell renewal systems such as intestinal epithelium and epidermis.


Synapses of optic nerve afferents in the rat suprachiasmatic nucleus. I. Identification, qualitative description, development and distribution. Synapses of optic nerve afferents (optic synapses) in the rat suprachiasmatic nucleus (SCN) have been identified ultrastructurally. They are easily distinguished from other types of synapses. The optic boutons are characterized by the presence of large mitochondria with a swollen electron lucent matrix and an interconnected tubular system formed by their inner membrane. Other, more variable features include: 1) a scattered pattern of synaptic vesicles which are found throughout the entire presynaptic element with relatively little accumulation near the active zones; 2) the occurrence of dense core vesicles and glycogen granules; 3) the active zones, the majority of which is Gray-type I, but a minority can obviously be classified as Gray's type II; 4) the innervation of smaller peripheral dendrites and dendritic spines. Boutons of this kind are exclusively filled with anterogradely transported horseradish peroxidase injected into both eyes. Very few neuronal elements containing the typical mitochondria have been observed in the SCN on the 6th day post partum, increasingly more on the 9th and 12th day, but considerably higher numbers after opening of the eyes on the 17th and the following days. The location of normal and degenerating optic boutons was examined light- and electron microscopically. In the rostral third of the SCN there are relatively few optic synapses which are found close to the optic chiasma. In the middle portion of the SCN optic synapses increase in number; they are found not only in the ventral part of the nucleus but also in lateral regions. This becomes particularly obvious in the caudal third of the SCN.


Occurrence of nuage in fetal human germ cells. Germ cells of human fetuses aged 7-13 weeks were studied. Dense fibrous material called nuage was found in the cytoplasm of human fetal germ cells. The nuage increased in frequency in the germ cells during the interval studied. In oogonia the nuage occurred much more frequently than in spermatogonia and the frequency of nuage in oogonia increased earlier than in spermatogonia. The number of nuage was continuously at a higher level in oogonia than in spermatogonia. It is suggested that these findings support the theory that the nuage may act as a germ cell determinant.


Unusual aggregations of tubules associated with endoplasmic reticulum in digestive cells of Alderia modesta (Mollusca: Gastropoda: Sacoglossa). Aggregations of cylindriform tubules closely packed in a hexagonal arrangement and associated with the rough-surfaced endoplasmic reticulum (RER) are described in cells of the digestive gland of a sacoglossan gastropod. Each tubule is approximately 61 nm in outer diameter and the central lumen is lined with a layer of finely granular material. The tubule wall is composed of approximately 22 dense subunits. Adjacent tubules are associated with a pair of indistinct granules. Membranes of the RER frequently join the aggregations. The lumen of a tubule may be continuous either with the intracisternal space of the RER or with the cytoplasmic matrix of the cell. It is suggested that the aggregations of tubules are elaborations of the endoplasmic reticulum.


Synapses of optic nerve afferents in the rat suprachiasmatic nucleus. II. Structural variability as revealed by morphometric examination. The identification of optic synapses in the rat suprachiasmatic nucleus (Güldner, 1978) has made it possible to study them morphometrically. The measurements followed the check-list introduced by Palay and Chan-Palay (1976). There are several items which could usefully be added to this list. The structure of essential synaptic components varies considerably in what is apparently one synaptic population based on morphological criteria. The possible reasons for the variable sizes of the optic boutons containing different amounts of clear and dense core vesicles are discussed in terms of different activities or metabolic states of the individual boutons and/or different metabolic states of neuronal and glial elements in their vicinity. The active zones of optic synapses are also extremely variable. One optic bouton can form several active zones of very different sizes, or form Gray-type-I ("asymmetric"), Gray-type-II ("symmetric") and intermediate contacts at the same time. The function and/or functional efficiency of a single optic bouton therefore could then be quite different with respect to its various postsynaptic elements. The different appearance of the active zones is discussed mainly in terms of possible regulative influences from neighboring synapses via the postsynaptic neuron.


Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea, Notostraca). Each ovarian follicle of Triops cancriformis is four-celled; these cells (one oocyte and three nurse cells) are interconnected by cytoplasmic bridges. In the course of differentiation, the nurse cells are clearly recognizable; they increase in size more than the oocyte and their nuclei contain many nucleoli. For the first time in Arthropoda, yolk globules are reported to be present in nurse cell cytoplasm; these globules arise from the smooth endoplasmic reticulum. The functional significance of the intercellular bridges and the trophic role of the nurse cells are discussed.


Surface functions during Mitosis I: phagocytosis, pinocytosis and mobility of surface-bound Con A. Phagocytosis, pinocytosis and the surface distribution of concanavalin A (ConA) have been analyzed during mitosis in several mammalian cell lines. Use of the bisbenzimidazole dye, Hoechst 33258, for chromosome staining after gentle fixation made possible the rapid identification and correlation of mitotic phase with surface properties. Phagocytosis of both opsonized and nonopsonized particles is markedly depressed in mitotic cells of the mouse macrophage cell line J774.1. The uptake of opsonized particles (IgG-coated erythrocytes) is impaired from early prophase through early G1, whereas phagocytosis of nonopsonized particles (latex beads) is restored by telophase. Fluid pinocytosis, determined by the uptake of soluble horseradish peroxidase, is also inhibited during mitosis. Thus peroxidase-containing cytoplasmic vesicles were virtually absent from mid-prophase through telophase in both J774 and Chinese hamster ovary (CHO) cells. Adsorptive pinocytosis of ConA was determined from the different distributions of fluorescence in single cells incubated at 37 degrees C with rhodamine-conjugated ConA (surface and cytoplasmic label), then fixed and further incubated with fluorescein-conjugated anti-ConA (surface only). The separate fluorescence of Hoechst, fluorescein and rhodamine could be optically isolated. In interphase J774 cells, ConA is rapidly internalized into cytoplasmic vesicles. In contrast, ConA is restricted to the plasma membrane from mid-prophase through telophase. In CHO, the depressed pattern of internalization is not fully established until metaphse. The surface distribution of ConA also varied dramatically as a function of mitotic phase. Between mid-prophase and early anaphase, the pattern of surface ConA-receptor complexes is diffuse. Once the cleavage furrow begins to develop, however, ConA moves into the region of the furrow. This was shown in J774, CHO and 3T3 mouse embryonic fibroblasts, and is probably universal. ConA movement into the membrane that overlies the microfilaments of the contractile ring is analogous to similar movements that occur in interphase cells during ConA cap formation and during the development of phagocytic pseudopods. The analogy emphasizes the common functional consequences of microfilament-membrane organization. It is evident that membrane processes which depend upon endocytosis--for example, certain hormone-induced signals--may be interrupted during mitosis. Inhibition of endocytosis thus may be a significant element in the control of cellular activities during mitosis and a strong influence on the properties of the emergent post-mitotic cell.


An estrogen receptor from Xenopus laevis liver possibly connected with vitellogenin synthesis. This paper describes an estrogen receptor which is found in both the nucleus and cytoplasm of liver cells from male Xenopus laevis, and which seems to be involved in the induction of vitellogenin synthesis. It has a high affinity for estradiol (Kd = 0.5 x 10(-9) M), and the affinities of various steroids for the receptor correlate well with their ability to induce vitellogenin synthesis. It sediments at 3.5S at 0 degrees C in 0.5 M KCI. The rate of sedimentation is unaffected by incubation at 20 degrees C prior to centrifugation, but increases if the salt concentration is lowered to 0.1 M KCI or to zero. It has a Stokes radius of 2.6 nm and a molecular weight of approximately 40,000. The receptor is present at very low levels compared to other steroid target tissues (50--100 fold less than chick oviduct). The cytoplasm of a single hepatocyte contains 92 +/- 18 binding sites for estradiol, while each nucleus contains 99 +/- 19 sites.


Premature of chromosome condensation in a ts DNA- mutant of BHK cells. A temperature-sensitive mutant of BHK, designated ts BN-2, shows a rapid drop in 3H-thymidine incorporation along with accumulation of the cells in the G1 phase of the cycle when asynchronous cultures are shifted from 33.5 degrees C to the nonpermissive temperature of 39.5 degrees C. Synchronized cultures of ts BN-2 cells did not enter DNA synthesis when shifted up in G1. Shift-up of cultures at the beginning of the S phase resulted in an approximately normal rate of DNA synthesis for about 2 hr. The rate of DNA synthesis then quickly declined, and the cells became arrested in mid-S after completion of approximately 0.5 rounds of DNA replication. At the same time, the majority of the cells were observed to lose the nuclear membrane and displayed premature chromosome condensation. These events were followed by the appearance of cells containing several micronuclei and eventual cell disruption and death. The nonpermissive temperature appeared to have no effect on either the elongation of short fragments of DNA or the execution of mitosis after the completion of the S phase under permissive conditions. The ts defect in this mutant may directly limit the initiation of DNA synthesis or alter the regulation of chromatin condensation.


The spliced structures of adenovirus 2 fiber message and the other late mRNAs. All late adenovirus 2 messenger RNAs, except perhaps that for peptide IX, are composite molecules with sequences derived from at least two to five or more separate portions of the genome, as determined by electron microscopic analyses of polysomal RNA hybridized to single- or double-stranded DNA. We previously reported that eight different rightward-transcribed mRNAs contain, at their 5' ends, a common three-part leader sequence derived from coordinates 16.6, 19.6 and 26.6, and that a ninth mRNA consists of sequences from 4.6--6.1 linked directly to those from 9.7--11.0 (Chow et al., 1977a; revised coordinates). We now demonstrate that four additional RNA species also have the same tripartite leader joined to messages with 5' ends located at coordinates at 29.1, 30.5, 33.9 and 42.8. The RNA transcript extending from 29.1--39.0 covers most or all of the viral associated (VA) RNA genes (29.0--30.1). The late RNAs can be grouped into several families of transcripts in which two or three mRNAs have the same 3' end and 3' proximal sequences, but differ substantially in length at the 5' end and, therefore, the coordinate at which the leader sequences are attached to the main body of the message. The number of RNA species transcribed from the Ad2 chromosome between coordinates 29 and 50 exceeds the number of identified Ad2 proteins. Several pairs of messages differing in length by a few percent may thus encode the same protein or have a precursor-product relationship. We have also found that a subpopulation of the polysomal mRNAs for the fiber protein, the most distal gene (86.3--91.2) in the rightward transcription unit, has a fourth leader component "y" (78.6--79.1) in addition to the common tripartite leader sequences. Less frequent fiber RNA species have fifth "x" (76.9--77.3) and/or sixth "z" (84.7--85.1) leader segments. Transcripts with x, y or z segments might be processing intermediates, and it is possible to arrange these species into an array suggestive of multiple pathways for the maturation of fiber mRNA. mRNA transcribed at late times from the L strand between coordinates 15.0 and 11.0 has a single component leader located 200 bases from the main body of the gene from coordinate 16.1 to 15.7. This leader originates from a site very close to the first leader component (16.5--16.6) of the R strand messages and suggests the presence of adjacent L and R strand promoters from which transcription diverges. Taken together, these results emphasize the diversity of splicing patterns and the variety of recombined sequences generated during the synthesis of late Ad2 mRNAs.


Template-determined, variable rate of RNA chain elongation. Q beta replicase polymerizes MDV-1 RNA at a markedly variable rate. Electrophoretic analyses of partially synthesized strands showed that a few of the elongation intermediates are much more abundant than others, reflecting a variable rate of chain elongation. Our data suggest that at a relatively small number of specific sites in the sequence of this RNA, the progress of the replicase is temporarily interrupted, and then resumes spontaneously, with a finite probability. Since the time spent between these pause sites is negligible compared with the time spent at pause sites, the mean time of chain elongation is well approximated by the sum of the mean times spent at each pause site. Nucleotide sequence analysis of the most prominent elongation intermediates indicated that they all have the potential to form a 3' terminal hairpin structure. This suggests that the marked variability in the rate of chain elongation is due to the formation of terminal hairpins in the product strand, or the reformation of hairpins in the template strand. A survey of the literature shows that this phenomenon occurs with most, if not all, nucleic acid polymerases. Structure-induced pauses may play a role in the regulation of nucleic acid synthesis.


Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes. The purine ribonucleoside triphosphate analogues adenosine 5'[gamma-S]triphosphate and guanosine 5'[gamma-T]triphosphate were used as affinity probes for studying RNA chain initiation in isolated nuclei from the mouse myeloma 66.2 cell line. Transcripts initiated with either nucleotide analogue were isolated by affinity chromatography on a mercury-agarose affinity column. The binding was specific and dependent upon the inclusion of the sulfur nucleotide analogues in the in vitro synthetic reaction. Several lines of evidence indicate that the affinity-labeled RNA is initiated in vitro. First, the sulfur nucleotide is recovered in high yield as a single nucleoside 5'[gamma-S]triphosphate 2',3'-monophosphate product following alkaline hydrolysis of RNA bound to the affinity column. Second, authentic ribosomal 5S RNA is known to initiate with GTP; in vitro 5S RNA is bound to mercury-agarose only if [gamma-S]GTP is used as the affinity label in the synthesis, and not if [gamma-S]ATP is used. Under the conditions studied, nuclei incorporated 1.2--2.4 pmole of UMP per 10(6) nuclei per min, and the rate of synthesis was unaffected by substitution of the nucleotide analogues for the normal nucleotides. The percentage of the total RNA synthesized that was incorporated into sequences initiated in vitro was 7.8 +/- 1.5% with [gamma-S]ATP and 9.6 +/- 6.4% with [gamma-S]GTP. The size of the total RNA synthesized, determined by sedimentation on sucrose density gradients containing dimethylsulfoxide, ranged from less than 5S to 45S, and the size of the affinity-labeled sequences ranged from less than 5S to 28S. Approximately half of the incorporation into RNA initiated in vitro was sensitive to a concentration of alpha-amanitin which selectively inhibits polymerase II activity. Most of the remaining incorporation into initiated sequences can be abolished by concentrations of alpha-amanitin that are inhibitory for polymerase III activity. Over 70% of the total incorporation into ribosomal 5S RNA transcripts was into sequences initiated in vitro. This initiation was catalyzed by polymerase III and was specific for GTP as the initiating nucleotide. The RNAase T1 fingerprint of the newly initiated 5S RNA indicates that this gene is accurately initiated and faithfully elongated in vitro. The use of these affinity label probes provides much greater sensitivity for studying the initiation of RNA in vitro.


Altered translatability of messenger RNA from suspended anchorage-dependent fibroblasts: reversal upon cell attachment to a surface. Anchorage-dependent cells, when forced into suspension culture, display a repertoire of dramatic, coordinated regulatory phenomena. Message production promptly decreases 5 gold but the cells maintain a constant amount of poly(A)+ by means of a concomitant stabilization of mRNA against decay. Protein synthesis shuts down much later and the mRNA is stored in a nonfunctioning state. In this study, the inactive mRNA is extracted from suspended cells and shown to have aberrant translation properties. Well defined polypeptides are apparently no longer synthesized when this mRNA directs protein formation in either reticulocyte or wheat germ-derived heterologous translation systems. Rather, shortened peptides are formed by this mRNA and these become smaller as mRNA is used from cells suspended for longer periods of time. Very few focused spots are formed when the aberrant polypeptides are analyzed in two-dimensional electrophoresis. The sedimentation properties of suspended cell mRNA and the size of poly(A) are unchanged from control monolayer cells. Cross-hybridization of cDNA transcribed from a control cell message population with suspended cell mRNA shows that all sequences are present in normal concentrations. While most identifiable spots disappear from the two-dimensional gel electropherograms of the protein products produced by suspended cell mRNA, a few polypeptides are still synthesized in relatively normal amounts. Conserved polypeptides are found in products of both the reticulocyte and wheat germ systems, but they are different products in each case. The lesion in the suspended cell mRNA does not seem to be at the 5' termini, since synthesis of the shortened peptides is fully sensitive to inhibition of pm7G. Cells that contain extensively modified message can resume protein synthesis when allowed to reattach to a solid substrate. There is an apparent remodification of mRNA to normal translatability within a few hours of cell reattachment, since mRNA from recovering cells quickly resumes directing relatively normal patterns of polypeptide synthesis in vitro. The restoration of normal message function occurs even when new message formation is blocked with actinomycin. Cells recovering after reattachment synthesize supranormal amounts of a few major proteins involved with cell structure, as shown in these studies by an increased amount of translatable sequences which encode these proteins. The most apparent enhanced message is that coding for actin. mRNA from recovering cells produces in vitro several times more actin relative to other proteins than does control cell mRNA. The enhancement of actin mRNA is not seen in the message population of cells that reattach in the presence of actinomycin. The results suggest a morphologically related induction of gene expression.


Evolutionary change in the repetition frequency of sea urchin DNA sequences. The frequency of occurrence of particular repetitive sequence families has been estimated in the DNA of the three sea urchin species Strongylocentrotus purpuratus. Strongylocentrotus franciscanus and Lytechinus pictus using individual cloned S. purpuratus repetitive sequence elements. Cloned repetitive sequence elements as described by Scheller et al. (1977a) were prepared by reassociation of S. purpuratus DNA fragments to repetitive Cot, digestion with single-strand-specific nuclease S1 and ligation of synthetic restriction sites to their ends. The sequences were cloned by insertion at the Eco RI site of plasmid RSF2124, labeled, strand-separated and reassociated with 800--900 nucleotide long unlabeled DNA. Both kinetic (genomic DNA excess) and saturation (cloned DNA excess) estimates of frequencies were made. For nine cloned fragments, the ratio of the repetition frequency in S. purpuratus DNA to that in S. franciscanus DNA ranges from about 20 to about 1. In the four cases examined, only a few copies were detected in the DNA of L. pictus. Estimates have also been made of frequency changes in many repetitive families by measuring the reassociation of labeled repetitive DNA fractions of each species with total DNA from other species. In each reciprocal comparison, the labeled repetitive sequences reassociate more slowly with DNA of other species than with DNA of the species from which they were prepared. Thus it appears that the dominant repetitive sequence families in the DNA of each species are present at lower frequencies in the DNA of closely related species. Measurements of thermal stability have been made of S. purpuratus cloned repetitive sequences reassociated with S. franciscanus DNA or S. purpuratus DNA. Most families have changed both in frequency and sequence, although some have changed little in sequence but show great changes in frequency.


Transcription of structural and intervening sequences in the ovalbumin gene and identification of potential ovalbumin mRNA precursors. Structural sequences that are extensively separated by nonstructural intervening sequences in the natural ovalbumin gene are coordinately expressed in target and nontarget tissue. The intervening sequences, which consist of unique sequences in the chick genome, are transcribed in their entirety. The amount of nuclear RNA corresponding to these sequences, however, is approximately 10 times less than that observed for structural sequences. The accumulation of RNA corresponding to structural and intervening sequences during acute estrogen stimulation suggests either that there are different rates of transcription for these regions of the ovalbumin gene or that RNA sequences corresponding to the intervening sequences are preferentially processed and degraded. Comparison of the in vitro expression of portions of the ovalbumin gene in nuclei isolated from chronically stimulated oviducts indicates that both structural and intervening sequences are preferentially transcribed in vitro at rates approximately 500 times greater than expected for random transcription of the haploid chick genome. In addition, electrophoresis of oviduct nuclear RNA on agarose gels containing methylmercury hydroxide reveals multiple species of RNA that are from 1.3 to over 4 times larger than ovalbumin mRNA and hybridize to both structural and intervening sequences of the ovalbumin gene. These results are consistent with transcription of the entire ovalbumin gene into a large precursor molecule followed by excision of the intervening sequences and appropriate ligation of the structural sequences to form the mature mRNA.


The isolation of structural genes from libraries of eucaryotic DNA. We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of beta-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.


Chloroplast membrane biogenesis in Chlamydomonas: correlation between the formation of membrane components and membrane structure. The y-1 mutant of Chlamydomonas reinhardi, when allowed to green in the presence of chloramphenicol (CAP), an inhibitor of protein synthesis on 70s ribosomes, form photosynthetic membranes which contain somewhat less chlorophyll than those of cells greened in the absence of the drug. Photosystem I and II activities are drastically reduced in the CAP-greened cells, and specific alterations in the polypeptide composition of the thylakoid membranes are also observed. We have examined the internal structure of the thylakoid membranes from cells greened in the presence and absence of CAP, and have found that the large particles observed on the exoplasmic fracture face (EF) are substantially reduced in size and number in the CAP-greened cells. This structural defect seems related to the absence of significant photo-system activities in the CAP-greened cells, despite the presence of most major membrane polypeptides. We suggest that CAP treatment results in a failure of the cell to organize functional reaction complexes, and is structurally reflected in the absence of large (EF) particles in such membranes. This defect can be repaired by allowing the affected cells to re-green in the absence of the drug, and the large particles reappear, paralleling an increase in photosynthetic activity.


Cellular resistance to fluoride.

Cellular resistance to fluoride. Cells cultured in vitro can be adapted to grow in the presence of high concentrations of fluoride by stepwise increasing the fluoride concentration in the culture medium. Such cells seem to have the ability to exclude fluoride from intracellular compartments.


Short duration of the mitotic and endomitotic cell cycle in the heterochromatin-rich monocot Allium carinatum. Fraction labeled mitoses curves and caffeine-induced binucleate cell populations, respectively, were used to determine the duration of the mitotic and endomitotic cycle in roots of Allium carinatum, a leak with a 2C DNA content of 22.4 pg and a high proportion of heterochromatin. The mitotic cycle lasts, on an average, 8.1 h, the endomitotic one 7.5 h. These relatively short times (with respect to the nuclear DNA content) are discussed in the view of a possible role of heterochromatin in shortening cell cycle duration and possible advantage of the endomitotic cycle for rapid cell and tissue growth.


Partial purification of centrioles from spleen cells. Isolation of a fraction enriched with centrioles from bovine spleen cells is described. The method involves preparation of a nuclear-centriolar complex and detachment of centrioles from the complex by mechanical treatment in a homogenizer. Purification of centrioles was achieved by centrifugation through a sucrose concentration gradient after treatment by ultrasound, DNase I and Triton X-100. The resulting centrioles retain their in situ ultrastructure.


Binding of the chemical carcinogen N-hydroxy-acetyl-aminofluorene to ploidy classes of rat liver nuclei as separated by velocity sedimentation at unit gravity. A large sedimentation device was developed that allows separation of 5 X 10(8) rat liver nuclei by velocity sedimentation at unit gravity. Using the apparatus isolated rat liver nuclei were separated into classes of diploid stromal (Von Kuppfer, sinusoidal lining) nuclei, diploid parenchymal nuclei and tetraploid parenchymal nuclei respectively. DNA content and volume of the nuclei were measured. Diploid nuclei were 100% pure; tetraploid nuclei 98%. The in vivo binding of the liver carcinogen [3H]-N-hydroxy-AAF to these classes of nuclei was determined (total binding to protein, DNA and RNA). Binding and the subsequent removal of the fluorene derivatives was registered as a function of time. At all stages diploid stromal nuclei bound 2.6--5 times less carcinogen than did diploid parenchymal nuclei. Tetraploid parenchymal nuclei bound more than twice (2.3--3.95) the amount, that was present in their diploid counterpart. This effect became more pronounced 11 days after application of N-hydroxy-N-acetyl-2-aminofluorene. DNA was enzymatically purified from pooled classes of the various nuclear types. For purified DNA also it was found that DNA derived from diploid stromal nuclei bound 2.6--2.8 times less carcinogen than did DNA derived from diploid parenchymal nuclei.


Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on lipid profiles in tissue of the Fischer rat. Female Fischer 344 rats were given single oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 10, 50 or 100 microgram/kg, and sacrificed 1, 3, 10, 14 or 21 days later. The fatty livers caused by a sub-lethal dose of TCDD involved a temporary increase in triglyceride and free fatty acid levels, with a persistent decrease in levels of sterol esters. In contrast, the fatty livers resulting from a lethal dose of TCDD involved a large increase in cholesterol esters and free fatty acids, with little change in triglyceride levels. These changes appeared to result in part from damage sustained by lysosomes. TCDD also altered the lipoprotein composition of the serum, the fatty acid composition of various lipid classes in liver and serum, and the ultrastructure of the liver (formation of myeloid bodies). A rapid, dose-dependent effect of TCDD, was the elevation of levels of organic-soluble fluorescent pigment in the heart. This pigment was found to match a previously characterized fraction of lipofuscins in fluorescence spectrum and chromatographic properties. The relatioship of these observations to a possible mechanism of toxicity for TCDD involving radical-induced lipid peroxidation is discussed.


Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture. In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 microgram/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 microgram/ml of 7,8 benzoflavone(BF). Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones. One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the +/-(trans)-7alpha, 8beta-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (+/-)-7alpha, 8beta-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydro-BaP (diol-epoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8. The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.


Comparison of in vivo and in vitro binding of polycyclic hydrocarbons to DNA. The in vivo binding of [3H]benzo(a)pyrene (BP) and 3-[3H]methylcholanthrene (3MC) to liver and lung DNA was studied in A/J mice. Only in liver was there any reduction in total DNA-bound radioactivity between 4 h and 24 h after administration of the hydrocarbon. DNA was fractionated on Sephadex LH-20 after enzymatic digestion. A single deoxyribonucleoside-BP adduct was detected whereas two major 3MC-adducts were observed. Wtih both BP and 3MC, three additional peaks of radioactivity eluted rapidly in the lung DNA experiments while a fourth was noted with liver DNA. The nucleoside-bound adducts from lung represented a much larger proportion of the total radioactivity than with liver. In vitro analysis of 3MC binding to DNA showed the nucleoside-bound adducts to be predominantly deoxyguanosine-dependent but that the early peaks were independent of base suggesting binding to another part of the DNA molecule, perhaps phosphate, i.e, phosphotriesters.


Metabolism of 35S-labelled copper-, zinc-and cadmium-thionein in the rat. A comparative study has been made of the metabolism in the rat of intravenously-administered hepatic copper-, zinc- and cadmium-thioneins. In all cases the 35S-labelled protein was rapidly removed from the circulation. About 20% of the 35S was present in the kidneys after 30 min byt only small amounts of 35S were found in the liver, intestinal mucosa or pancreas. In the case of copper-thionein, 30% of the injected 35S was recovered in the urine within 2 h, mainly as intact copper-thionein. The 35S which appeared in the kidneys was also present initially as metallothionein but this was degraded very rapidly, especially when zinc-thionein was give. Both copper and cadmium from the injected proteins accumulated in the kidneys as metallothionein, but there was no increase in renal zinc concentrations in the rats dosed with zinc-thionein. These findings are discussed in relation to the development of renal damage in chronic cadmium and copper toxicity.


Evidence for alteration of the membrane-bound ribosomes in Micrococcus luteus cells exposed to lead. Micrococcus luteus cell exposed to PB(NO3)2 contained cytosol ribosomal particles and disaggregated membranal ribosomal particles as determined by ultracentirifugation and spectral studies. Approx. 60% of the membrane ribosome fraction from lead exposed cells had a sedimentation value of 8.4S. Cytosol ribosomes from lead exposed cells as well as membranal and cytosol ribosomes from control cells were comparable by their contents of predominantly the 70S type with the 50S and 100S present in relatively small amounts. The lead content of the 8.4S component was more than 200 times higher than the components with higher sedimentation coefficients from lead exposed cells and approc. 650 times more than that of control cell ribosomes. The cells exposed to lead, however, showed no adverse effects from the lead in respect to their growth rates and cellular yields. These results indicate that lead is interacting only at specific sites of the membrane and is inducing events initiated only in strategic cellular regions. These data further substantiate that subtle changes do occur in lead exposed cells that show no obvious effects. It is assumed that these 'minor' alterations are, in toto, biologically significant.


Monolayers of sterols and phosphatidylcholines containing a 20-carbon chain. Pressure-area curves of monolayer films were measured for phosphatidylcholines (PC) in which the 1-position was occupied by palmitic acid and the 2-positions were occupied respectively by: 20 : 0,20 : 1n9, 20 : 2n6, 20 : 3n3, 20 :3n6, 20 :3n9, 20 :4n6 or 20 : 5n3 fatty acids. The interactions of these PC with cholesterol or desmosterol were studied. Fully saturated PC (16 : 0--20 : 0) displayed a relatively small molecular area. The presence of one double bond greatly increased the molecular area, but a second double bond resulted in only a small additional increase in area. A third double bond caused a further large expansion in area, but the presence of a fourth or fifth double bond had little additional effect. Condensation of molecular area was observed with all sterol/PC mixed films. Approximately equimolar mixtures of sterols and unsaturated PC condensed maximally, but 16 : 0--20 : 0 PC condensed most in mixtures containing 20--30 mol% of either sterol. The extent of condensation varied with surface pressure. The pressure at which maximum condensation occurred depended upon the structure of the PC and was always 20 dyn/cm or lower. The pressure at which maximum condensation with cholesterol occurred increased with increasing unsaturation of the PC.


Temperature dependent ionic structure of phospholipid monolayers. Radiotracer studies of calcium adsorption to dipalmitoylphosphatidyl-alkanolamine monolayers measured at various temperatures showed that the binding constant of calcium increased with temperature up to around 30 degrees C but the decreased on exceeding this critical temperature. The temperature dependent ionic structure of ampholytic phospholipid monolayers are discussed.


The solubilization of progesterone by mixed bile salt-phospholipid sols. The solubility of progesterone was determined in several different bile salt-phospholipid mixtures, and it is concluded that: (1) The solubility in unconjugated bile salts is greater than in the conjugated analogues, and the solubility in deoxycholate solutions is twice that in cholate solutions. (2) Substitution of hydroxyl groups in the 11 and 21 positions of progesterone increases solubility, whilst substitution in the 17-position decreases solubility in bile salt solutions. (3) Progesterone solubility in mixed bile salt solutions is proportional to the mole ratio of the surfactant mixture. (4) Sodium deoxycholate (SDC)-phospholipid sols show no such linear solubilizing properties; a minimum occurring at a mole ratio of SDC to phospholipid of 1 : 4. (5) There is a break in the solubility curve of progesterone in lysophosphatidycholine (LPC)/phosphatidylcholine (PC) mixtures at a mole ratio of 65 : 35 coincident with maximum viscosity. (6) Introduction of SDC into LPC/PC mixtures results in decreased progesterone solubility.


Differential scanning calorimetry studies of some analogs for the lipid component of biological membranes. The melting behavior of members of newly synthesized series of rac-1,2-diglycerides with substituted phenyl groups or a benzyl group on the 3-position was investigated with differential scanning calorimetry (DSC). Solution crystallized samples had single melting temperatures, higher than those of the quenched or annealed specimens. Quenched samples exhibited polymorphic behavior; some melted and recrystallized during slow heating. This behavior is similar to that of lecithins and suggests that X-ray diffraction studies of the substituted diglycerides may be useful for understanding membrane structure and functions.


The effect of free fatty acids on the thermotropic phase transition of dimyristoyl glycerophosphocholine. The effect of free fatty acids on the phase transition characteristics and fluidity of bilayers of dimyristoyl glycerophosphocholine were studied by pyrene eximer fluorescence and differential scanning calorimetry. High melting saturated fatty acids with chain lenghts of 12--18 carbon atoms raise the phase transition temperature and enhance the ability of pyrene to form clusters in the gel state while not affecting the fluidity of the membrane in the liquid crystal state. Low melting unsaturated fatty acids lower the phase transition temperature and decrease the ability of pyrene to form clusters in the gel state while not affecting the fluidity of the membrane in the liquid crystal state. The effects of the very long chain fatty acids, arachidic (C 20) and behenic (C 22) appears to be similar to those of cholesterol in that they cause a broadening of the phase transition with a lowering of the transition enthalpy but have little effect on the temperature at which the phase transition occurs.


Raman spectroscopic studies of biological membrane model systems: dietherlecithins. Raman spectra are obtained for the multilayer dispersion of rac-1,2,dioctadec-9'-cis-enyl-glycero-3-phosphorylcholine (dietherlecithin) in excess water. The CH stretching region was studied as a function of temperature and indicates that the multilayer dispersions undergo a liquid crystal to the gel phase transition at--21 +/- 4 degrees C.


The hydration of phospholipids and phospholipid-cholesterol complexes. The hydration characteristics of phosphatidylcholines and the effect of cholesterol on these were studied with differential thermal analysis and water vapour adsorption experiments. Also the water adsorption of egg phosphatidylethanolamine and the effect of cholesterol on this was studied and compared with corresponding qualities of phosphatidylcholine. The differential thermal analysis study showed that the monohydrates of egg, dipalmitoyl, and dioleoyl phosphatidylcholine tightly bind approximately 9 molecules of water per phosphatidylcholine molecule. Cholesterol is proved to somewhat increase the water binding of the phospholipids. Cholesterol is also shown to decrease the heat change of the chain melting transition of dioleoyl phosphatidylcholine, but not to abolish it completely. The water adsorption experiments indicate that the hydration of phosphatidylcholines takes place in two steps; a strong initial water binding and a second phase of weak binding. The adsorption isotherm of egg phosphatidylethanolamine is strikingly different from that of egg phosphatidylcholine. Cholesterol is shown, also by this method, to increase the hydration of phospholipids especially that of dipalmitoylphosphatidylcholine. The results in this study are in good agreement with those presented by many other authors. Starting with the accumulated information of the hydration characteristics of phosphatidylcholines the organization of the bound water around the polar group is discussed and the most probable model is evaluated.


[Animal study on radioactive antigenic fractions isolated from V.cholerae. I. The Ch 1 + 2 fraction]. The Ch 1 + 2 fraction has been marked by means of culture of Vibrio cholerae Ogawa HK1 on a synthetic medium containing leucine H3. The antigen distribution has been then studied before and after vaccination with the same non-marked antigen in normal and axenic mice, at the same time by the demonstration of radioactivity and radioimmunofluorescence in intestine, spleen, liver, kidney and thymus. Whichever the administration route, intestine and spleen are first stimulated, then more intensively with the second injection or ingestion. When administrated per os the fraction crosses the intestinal barrier and is fixed on the main lymphoid organs: intestine, spleen, thymus. After ingestion or injection into axenic mice, spleen is the first organ stimulated.


[Variation of helminthiasis incidence in Reunion as a function of different parameters]. Four years after Picot's experiments on parasitic diseases in Réunion Island, and after a large sanitation campaign, the authors valued the effect of this campaign on parasitic incidence. They showed that it had significantly decreased. At the same time, they pointed out the leading part of water quality in parasitic incidence; particularly, they showed that there are twice less Ascaridiasis when water is bacteriologically and chemically safe.


On the biosynthesis of guanidinosuccinate. We report a study motivated by a report that guanidinosuccinate is formed by transamidination from arginine to aspartate by perfused liver [J. Clin. Invest 57, 807 (1976)]. We prepared viable liver cells and incubated them with [14C]arginine labeled at the guanidino carbon and aspartate labeled at the methylene groups with tritium. A diacetyl-reacting band, similar to that reported with the perfusate in the above reference, was obtained by column chromatography. This band did not give a Sakaguchi reaction and contained no measurable tritium or 14C. Thus it did not derive from aspartate or arginine. On electrophoresis at pH 5.0, the diacetyl-reacting material moves to the cathode, guanidinosuccinate to the anode. The absorption spectrum of the diacetyl-reacting band showed a double peak, with maxima at 539 and 432 nm; guanidinosuccinate has only one maximum, at 533 nm. The diacetyl reagent reacts with sulfhydryl compounds and polypyrroles (e.g., bilirubin) to produce blue colors with significant absorbance in the 432-nm range. We saw no evidence for guanidinosuccinate formation by transamidination in these experiments with viable liver cells.


[Use of Cetremide in the surgery of hydatid cysts]. The satisfactory treatment for hydatid disease is surgical removal of the cyst. The use of different chemical scolicide such as formalin, alcohol and hypertonic solution of NaCl intra-cavity are common. The purpose of this paper is to present the use of a new scolicide agent CETRIMIDE (cetyl trimethyl ammonium bromide), first experimentally then in 7 patients with hydatid disease in different location. The special techniques described, no sign of toxicity or recurrence noticed. I have found this is a reliable method of preventing recurrences. No recurrence was found in our series after 2 years of follow-up.


Automated development of a kinetic method for the continuous-flow determination of creatinine. A "stopped-flow" method for the kinetic Jaffé determination of creatinine was developed, with the use of a computer-controlled continuous-flow system. Simplex optimization was used to find conditions of hydroxide and picrate giving maximum sensitivity for creatinine. We used a modified central composite experimental design to evaluate creatinine sensitivity and albumin, glucose, and acetone interferences as functions of hydroxide and picrate concentrations. More importantly, this work illustrates that the automated development of clinical chemical methods offers an efficient means of obtaining optimized, well-understood analytical procedures for subsequent routine use in the clinical chemistry laboratory.


Similarities of human hydatid cyst fluid components and the host serum. Nine lung hydatid cyst fluid of Echinococcus granulosus species from man were analysed by electrophoresis, immunoelectrophoresis and by biochemical tests. In addition respective sera of the host were analysed for comparison. Analysis revealed striking similarities in cellulose acetate and agarose gel electro- and immunoelectrophoretic patterns of proteins from hydatid cyst fluid and the serum of the respective hosts. It is presumed that serum proteins (albumin and globulins) occur in hydatid cyst fluid, but in smaller amount than in serum, an we believe that the host proteins can penetrate the membranes of the hydatid cyst.


Evaluation of a new multichannel analyzer, "Astra-8". We evaluated a new multiple-channel chemistry analyzer, the Beckman "Astra-8," which is controlled by a microprocessor with 52K bytes of memory. The instrument we tested performed the following tests: Na, K, CO2, chloride, urea nitrogen, glucose, and creatinine. We compared the Astra-8 to a continuous-flow (SMA 6/60) and a discrete (Du Pont aca, urea nitrogen only) analyzer. The Astra-8 demonstrated excellent precision, linearity, accuracy, analytical recovery, lack of interference, ease of operation, and satisfactory comparison to values obtained by the comparison methods.


Developmental patterns of visually handicapped children. This article reports a comparative study of the developmental patterns of blind, partially sighted and fully sighted children during the first few years of life. Using the (previous reported) Reynell-Zinkin developmental scale for visually handicapped children, the groups were compared in the five developmental areas of (a) social adaptation, (b) sensori-motor understanding, (c) exploration of environment, (d) verbal comprehension and (e) expressive language. It was found that 10 to 12 months was the age at which the sighted group began to outstrip the visually handicapped children in most of the developmental areas. This divergence increased until towards, the upper end of scale (4 to 5 years for the visually handicapped) when more abstract thought processes began to develop. The effect of visual handicap was also seen in the greater advantage of the partially sighted over the blind children. The patterns of development varied somewhat in the five different areas. The reasons for this are discussed. The findings point to the need for intensive help for visually handicapped children from the earliest months of life.


Improved liquid chromatographic determination of placental estriol in urine. Placental estriol in urine can be determined, after enzymatic hydrolysis, by liquid chromatography. A single-step extraction technique results in rapid processing with minimal sample manipulation. Analytical recovery of estriol added to urine was 96 to 106%. Day-to-day precision (CV), established by 10 replicate analyses at a mean concentration of 15.9 mg of estriol per liter, was 3.7%.


Interlaboratory comparison of results of erythrocyte protoporphyrin analysis. Each of 65 laboratories analyzed 10 whole-blood samples for erythrocyte protoporphyrin by one or more of several analytical procedures. These procedures were of two types: (a) extraction of protoporphyrin from the erythrocytes into ethyl acetate/acetic acid, re-extraction into hydrochloric acid, and fluorometric measurement; or (b) direct reading in a portable fluorometer (hematofluorometer), with no pretreatment of the blood sample. Interlaboratory correlation was generally poor, especially between laboratories using extraction procedures. Hematofluorometric results intercorrelated better, but they had a low bias as compared to the extraction approach. Nationwide standardization of the test is required to assure satisfactory interlaboratory performance and to identify laboratories whose results are sufficiently accurate to be used for interpretations according to guidelines set forth by the Center for Disease Control for erythrocyte protoporphyrin testing.


Occupational commitments and paternal deprivation. In the post-war era the husband-father role has been enlarged to encompass active participation in family life. However, occupational commitments may inhibit active involvement in family life, particularly in the middle classes where work has traditionally been defined as salient. This article reports on an investigation of the consequences of heavy work commitments for father role performance and child development. This investigation, using the case study method, focussed on hospital medicine and the early child-rearing stage of the family life cycle. It was found that fathers felt themselves to be shadowy figures in the background of their children's lives and that mothers experienced single-handed responsibility for child-rearing as burdensome. Children have little experience of a father's companionship, and discipline and work pressures may be transmitted to them. A few children exhibited behaviour difficulties. Suggestions for further study are made.


Considerations in the assessment of blind children who function as severely or profoundly retarded. Unevenness in development of blind children who function as severely or profoundly retarded, particularly in language and communication, needs to be considered as a feature of the child's ability to learn rather than as characteristics of his retardation. Impact of blindness on early development, environment and previous opportunities for learning need to be considered as part of the assessment process as well as of education and treatment.


An enzymatic radioassay for gentamicin.

An enzymatic radioassay for gentamicin. An enzymatic, radiochemical assay procedure for measuring serum gentamicin by use of gentamicin 3-acetyl-transferase and [acetyl-14C]acetyl-Coenzyme A is described and evaluated. The enzyme stoichiometrically and quantitatively transfers a radioactive label to the analyte during a 10-min incubation at 37 degrees C. The labeled gentamicin is then absorbed onto phosphocellulose paper discs, which are washed to remove unreacted [acetyl-14C]-acetyl-Coenzyme A and counted in a liquid scintillation counter for 1 min each. The assay detects gentamicin in concentrations as low as 0.2 mg/liter and gives a linear response to concentrations as high as 20 mg/liter. Sisomicin, a structural analog of gentamicin, is measured by the procedure, and tobramycin and netilmicin are slightly reactive. No other interferents were found among other aminoglycosides, other antibiotics, or substances endogenous to serum. Results by the new method are compared to those by radioimmunoassay and a microbiological method.


Atomic absorption spectrophotometry of chromium in serum and urine with a modified Perkin-Elmer 603 atomic absorption spectrophotometer. Modification of a Perkin-Elmer 603 atomic absorption spectrophotometer by adding a high-intensity tungsten-halogen lamp for background correction significantly improved the detection limit for elements that have analytical wavelengths in the near-ultraviolet and visible regions. Chromium in human serum and urine can be measured, with a simplified sample-handling technique, in concentrations of less than 0.1 microgram/liter. For comparison, the mean value for chromium in the serum of eight men was 0.14 microgram/liter.


Radioimmunoassay for urinary lysozyme in human serum from leukemic patients. We present a radioimmunoassay for lysozyme in human serum, based upon human lysozyme isolated from the urine of leukemic patients and antiserum prepared against this lysozyme in the goat. In the separation step, a second antibody is used. By properly adjusting the concentrations of unlabeled and 125I-labeled lysozyme and of the antibodies, maximal precision (SD, 0.04 mg/litre) was obtained in the range 0.00 to 2.00 mg/litre. In 20 normal volunteers the lysozyme concentration was 4.6 +/- 0.8 mg/litre (mean +/- SD), in 13 patients with monocytic leukemia 34.4 +/- 8.6 mg/litre. Correlation with lysoplate determinations was excellent in leukemic sera (r = 0.97) but was poor in normal sera (r = 0.35), possibly owing to the existence of isoenzymes.


The 4-hydroxybenzoate/4-aminophenazone chromogenic system used in the enzymic determination of serum cholesterol. A single reagent, containing cholesterol oxidase, cholesterol esterase, peroxidase, 4-hydroxybenzoate, and 4-aminophenazone, is used in determining serum cholesterol. Analysis time is 15 min, and the standard curve is linear to 6.0 g/liter. Analytical recovery of cholesterol was 100.1 +/- 0.4%. Within-run precision (CV) was less than or equal to 1.4 1.4%, between-run less than or equal to 4.8%. Comparison with results by a Liebermann Burchard method [Clin. Chim. Acta 5, 637 (1960)] gave a linear regression of y = 1.08x--0.05, with a correlation coefficient (r) of 0.985. Comparison with the Roeschlau enzymic method [J. Clin. Chem. Clin. Biochem, 12, 226 (1974)] gave y = 1.02x + 0.01 (r = 0.958). Comparison with the enzymic method of Allain et al. [Clin. Chem. 20, 470 (1974)] gave y = 1.01x--0.00 (r = 0.995). The following substances do not interfere up to the indicated concentrations (mg/liter): hemoglobin (5000), bilirubin (100), reduced glutathione (150), l-cysteine (400), urea (3000), creatinine (200), uric acid (200), d-glucose (10000), L-ascorbic acid (50), acetylsalicylic acid (500), L-DOPA (10), ergothioneine (1000), 2,5-dihydroxybenzoic acid (20), and 3,4-dihydroxybenzoic acid (10). Stored in an amber-colored bottle, the working reagent is stable for three months at 2--8 degrees C and for three weeks at 25 degrees C.


High-performance liquid chromatography of quinidine in plasma, with use of a microparticulate silica column. We describe a routine method for determining plasma concentrations of quinidine by liquid chromatography. The procedure requires 1.0 ml of plasma (or serum) and involves internal standard addition, extraction with ether, and separation on a column of microparticulate silica. Day-to-day CV (15 days) was less than 5% and no deterioration in column performance has been observed during 12 months. Comparison with a fluorometric procedure gave a correlation coefficient of 0.995.


Modified micro-scale enzymatic method for plasma ammonia in newborn and pediatric patients; comparison with a modified cation-exchange procedure. We modified two "kit" procedures for plasma ammonia to make them suitable for use with newborn and pediatric patients. Available methods either require too large a specimen, are insufficiently sensitive in the normal range, or are too cumbersome. One was an enzymatic (Sigma Chemical Co., St. Louis, Mo. 63178), the other a cation-exchange (Hyland, Costa Mesa, Calif. 92626) method. The modified micro-scale enzymatic method requires 100 microliter of plasma (CV at 38 micronmol/liter, 8%). Reagent costs per patient assay, including controls and standards, are currently $0.53. An assay run can be completed in 30 min. The modified cation-exchange method requires 200 microliter of plasma; the within-run CV is 12% for a concentration of 34.2 micronmol/liter. Reagents currently cost $0.74 per assay and each run requires less than 2 h to complete. The correlation coefficient for the two methods was 0.98.


Non-chromatographic screening test for hyperprolinemia. This procedure for determining serum proline in patients with hyperprolinemia involves protein precipitation (Folin-Wu method),  color development with isatin, extraction of the color with methylene chloride and measurement of its absorbance at 600 nm. The specificity and analytical recovery show the method to be suitable for this use.


Analytical precautions in measurement of blood cyanide. Solutions of KCN in 0.1 mol/liter NaOH, stored for six months at 4 degrees C, were unchanged as determined by silver titration and color development with pyridine-pyrazalone reagent. At room temperature the cyanide concentration of such solutions decreased by 0.079% per day (half life = 880 days) and the extent of color development changed in direct proportion to the change in silver titer. Rubber stoppers adsorb (or dissolve) HCN when in contact with this gas. The cyanide tends to come out of the stopper long after the source of the gas is removed. Rubber stoppers and expired air contaminated by HCN must be carefully avoided during cyanide analysis. Expired air contains HCN, generally in larger amounts for persons who smoke.


Microchromatographic methods for hemoglobin A2 quantitation compared. On 20 consecutive work days during four weeks, one technologist performed 24 microchromatographic determinations of hemoglobin A2 (Hb A2) by each of four methods: the Efremov procedure requiring Tris/HCl buffer, the original Huisman technique with use of glycine developer, and two commercial test kits in which a modified glycine developer is used. The bloood samples tested were obtained from 12 adults with no hematological abnormality and from 12 beta-thalassemia carriers previously diagnosed by familial and hematologic studies. Results by the first method and the two commercial kits (one from Helena Laboratories and one from Isolab, Inc.) did not differ significantly in precision for either the normal or beta-thalassemia trait samples. For both sample types, the second method yielded larger coefficients of variation than those obtained with the other methods. Moreover, the second method was the only one with which values overlapped for normal samples and samples with above-normal Hb A2 concentrations.


Kinetic centrifugal analyzer and manual determination of serum urea nitrogen, with use of o-phthaldialdehyde reagent. o-Phthaldialdehdye reagent was used in assaying urea with a miniature centrifugal analyzer. Seventeen samples can be assayed kinetically in 2 min at a current cost of less than a penny per test. The reaction is initiated in the instrument upon simultaneous mixing of the o-phthaldialdehyde reagent containing 10 microliter of sample with dilute sulfuric acid/N-(1-naphthyl)ethylenediamine dihydrochloride reagent. The reaction is monitored kinetically at 546 nm during the initial 2 min, when the rate is nearly constant. The reaction of o-phthaldialdehyde with urea reaches equilibrium by about 7 or 8 min, but the method cannot be used for end-point analysis because of interference by the blank reaction. A 1.2 g/liter solution of o-phthaldialdehyde used in conjunction with 2 mol/liter sulfuric acid solution was found to be most suitable for centrifugal analyzers. I also describe a manual version of the procedure that gives a linear standard curve up to 1.0 g of urea nitrogen per liter.


[Cholinesterase, arylesterase and proteinuria. A clinical trial in glomerular and tubular proteinuria (author's transl)]. A study is presented on the activity of cholinesterase (substrate acetylcholine) and of arylesterase (substrate phenylactate) in proteinuria, classified according to the results of electrophoresis of glomerular and tubular proteinuria. Comparison is made with the corresponding serum. The urine is concentrated by dialysis on polyethylene-glycol to 60 g protein per 1000 before determination of the activities. In the presence of equal quantities of protein, cholinesterase is slightly more active in glomerular than in tubular proteinuria. In selective glomerular proteinuria, cholinesterase and arylesterase are less active than in cases with little or no selectivity. Comparison with serum, in each individual case, indicates that the ratio of activity in concentrated urine to that in serum, is higher for cholinesterase and arylesterase in tubular than in glomerular cases, whereas the reverse is true in urine at its natural concentration, on account of the lower degree of proteinuria in tubular cases. The ratio of cholinesterase to arylesterase activity in concentrated urine and serum is determined. Since cholinesterase activity is greatly increased in glomerular cases (nephrosis) this ratio is on average markedly higher in glomerular proteinuria than in serum, whereas it is similar in urine and serum of tubular cases. These results, seen in the light of the molecular weights of the enzymes, are difficult to interpret with certainty, especially as regards tubular proteinuria.


[Determination of normal levels of urinary xanthurenic acid as a function of age and sex (author's transl)]. A quantitative determination of xanthurenic acid in human urine has made possible a statistical study of the secretion of this substance according to age and sex.


Thin-layer isoelectric focusing of hemoglobin variants: screening and determination of isoelectric points. The high resolving power of thin-layer isoelectric focusing was applied for screening some hemoglobin variants classified on the basis of their electrophoretic mobility in: electrophoretically slow variants (as Hb A2), electrophoretically slow variants (as Hb S), electrophorectically fast variants (Hbs type J). An analysis of the variant compounds has been performed, and the corresponding pI values were determined in whole hemolysate.


A method for measuring hydroxylysine and glycosylated hydroxylysines in urine and protein hydrolysates. A method for measuring hydroxylysine and glycosylated hydroxylysines is described, based on the separation of the 3 compounds by ion-exchange chromatography, followed by spectrophotometric analysis of the hydroxylysine present as glucosylgalactosylhydroxylysine, galactosylhydroxylysine and free hydroxylysine. The method does not require prior preparation of the urine sample nor the use of high-resolution ion-exchange systems. The method is applicable to the determination of the glycosylated hydroxylysine and hydroxylysine content of urine or alkaline hydrolysates of proteins.


Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. A new colorimetric method for quantitative analysis of serum lipid peroxide, free of interference from sialic acids, has been developed. We have used the thiobarbituric acid dissolved in sodium sulfate solution and both liberation of lipid peroxide and color reaction have been performed simultaneously by heating serum protein precipitate with this reagent in a weak acid solution. The new method is specific and facilitates the precise measurements of serum lipid peroxide. The average values determined by the new method increased slightly with age in healthy subjects. In patients with sequelae of cerebrovascular disorders, serum lipid peroxide values were higher than in healthy controls. These results may demonstrate the important role of lipid peroxide in aging and cerebrovascular disorders.


Middle-sized ninhydrin-positive molecules in uraemic patients treated by repeated haemodialysis. II. Chief peptide constituents of the faction. The fraction of middle-sized molecules obtained from haemodialysate by chromatography on Dowex 50W x 8 resolves upon thin-layer electrophoresis on Sephadex G-25 into nine ninhydrin-positive fractions and one staining only with tolidine reagent. All ten electrophoretic fractions give similar filtration profiles with Bio-Gel P-6 in the presence of SDS and 2-mercaptoethanol. The chief peptide components common to each electrophoretic fraction have molecular weight approx. 3000 and are especially abundant in lysine, glycine, glutamine acid and serine, while aromatic amino acids are in very low concentration.


Interrelationship between vitamins E and A: a clinical study. Plasma vitamin A and vitamin E levels were determined in 45 children. Seven normal children and 7 children with vitamin A deficiency were given daily supplements of 100 mg vitamin E for two weeks. Seven others received a placebo and served as controls. The mean levels of plasma vitamin E and A were 694 microgram/dl and 21 microgram/dl respectively. There was no correlation between plasma levels of the two vitamins. Administration of vitamin E resulted in a significant increase in plasma vitamin A concentration both in normal children and in those with vitamin A deficiency, while there was no change in the control group.


On the iron content of human serum ferritin, especially in acute viral hepaptitis and iron overload. A new method has been developed to measure ferritin iron in human serum. The ferritin is bound to antibodies to ferritin, which are coupled to Sepharose 4-B and separated from the serum by centrifugation. The iron is liberated from the bound ferritin and measured colorimetrically. The detection limit of this method proved to be approx. 15 ng Fe/ml serum. The serum ferritin iron concentration has been compared with the serum ferritin protein concentration. During liver disease with liver cell leakage the mean iron content of serum ferritin proved to be less than the mean iron content in the liver ferritin. The mean iron content in the liver ferritin was 16%, range 7%--32% (n = 8). The mean iron content in serum ferritin from the same patients was 5%, range 0%--14%. Also in two cases of haemosiderosis the serum ferritin iron content was low. It is suggested that ferritin loses part of its iron on passage from the tissue cells to the blood. In some cases of severe liver cell (or other cell) leakage the mean iron content in serum ferritin might be high, because then more or less complete ferritin molecules enter the blood.


Study of influence of sex and age on human serum lysosomal enzymes by using 4-methylumbelliferyl substrates. The sex and age dependence of activity of eight glycosidases and acid phosphatase was assayed in serum samples using the 4-methylumbelliferyl substrates. The activity of these enzymes does not change in relation to sex and to age except for acid phosphatase and beta-galactosidase which show significantly higher values in children as compared to adults. The usefulness of the 4-MU substrates for the detection of homozygotes for those lysosomal diseases involving one of the glycosidases studied is discussed.


Influence of endogenous S-adenosylmethionine on the determination of catechol O-methyltransferase activity in red blood cells. A double isotope method for the simultaneous determination of S-adenosylmethionine (SAM) and catechol O-methyltransferase (COMT) activity in red blood cells was developed. Healthy persons contained 7.9 +/- 1.9 nmol SAM per ml red blood cells and 1.9 to 3.8 nmol per ml plasma. Epinephrine increases the SAM biosynthesis rate in red blood cells. The possible influence of endogenous SAM on previous methods to determine COMT activity in red blood cells is discussed. The statement of Briggs and Briggs (Briggs, M.H. and Briggs, M. (1973) Experientia 29, 278--281) that COMT activity in red blood cells is lower during the last 3 months of pregnancy could not be verified by us.


Serum and urine electrolytes in primigravid Africans during pregnancy and postnatal period. Serum and urine electrolyte changes were studied in 55 primigravid African women during pregnancy and the postnatal period. The findings suggest that there is a fall in the serum sodium in the first trimester, which is followed by a gradual and steady rise throughout pregnancy. The serum potassium did not show any consistent pattern, while the serum chloride generally followed that of the serum sodium. These findings are similar to those reported for the Caucasian, although the African subject displays a lower serum sodium level and a much higher chloride level throughout pregnancy and the postnatal period. It is suggested that the reduction in sodium excretion noted in the third trimester is a reflection of the increasing sodium concentration in the blood with advancing pregnancy. The few patients who developed oedema and pre-eclampsia excreted relatively larger amounts of sodium in the puerperium.


Improvement of the FeCl3 test for phenylpyruvic acid. A simple alternative colorimetric method has been developed for quantitative determination of phenylpyruvic acid in urine based on the improved stability of the ferric complex of enol-phenylpyruvate in water-dimethylsulfoxide 50% vol. mixed solvent. The method can be applied directly to urine or to a dry ether-extract of urine. Operating on the dry ether-extract of urine, detection of phenylpyruvic acid is possible at an amount of as low as 1 mg/100 ml. Whereas, if urine samples are processed, detection is possible for a concentration of as low as 5 mg/100 ml and quantitative determination for a concentration greater than 10 mg/100 ml. The test carried out on a diaper wet with urine retains the same sensitivity and shows instantaneous development of the green colour.


The apoenzyme of aspartate aminotransferase and alanine aminotransferase in the serum of healthy persons and patients suffering from liver diseases. In activity determination with addition of pyridoxal 5'-phosphate (P-5-P), aspartate aminotransferase (AST) activity increases by 6.5 U/l and that of alanine aminotransferase (ALT) by 2.5 U/l in the serum of healthy persons. This corresponds to a relative stimulation of initial activity by 37% and 15.2%, respectively. ApoAST activity in patients with chronic liver diseases is not changed as compared with that of healthy persons, the relative stimulation rate, however, is significantly smaller. ApoALT activity and corresponding relative stimulation is significantly greater as compared with healthy persons. In the case of acute viral hepatitis, a decrease of AST and ALT activity is followed by a decrease of apoenzyme activity in the course of disease. Diagnostic evidence of determinations of aminotransferase activities could not be improved by addition of P-5-P.


Nucleic acid metabolism disturbance in progressive systemic sclerosis (systemic scleroderma). The content of helical forms of RNA and DNA in plasma was determined in 28 patients with progressive systemic sclerosis (PSS). Various disturbances of nucleic acid (NA) metabolism were noted in those patients: the DNA level was increased in comparison with the standard, and the RNA level was decreased. During treatment a normalization of the given indices was observed. The DNA content in plasma of PSS patients correlated with activity of the process, RNA content with the course of the disease.


Combined enzymatic assays for 3-hydroxybutyrate and lactate: end-point and kinetic methods. The consecutive measurement of 3-hydroxybutyrate and lactate by enzymatic end-point and rapid kinetic methods is described. Using end-point analysis within batch coefficient of variation (C.V.) ranged from 0.5 to 6.5% depending on concentration, and between batch C.V. was 1.7 to 9.0%. In the rapid kinetic method C.V. ranged from 4.2 to 10.6%. Both assays showed absolute agreement with conventional spectrophotometric methods. The rapid kinetic method yields plasma values within 15 min of blood sampling and is suitable for use in emergency situations.


Organic acid excretion in a patient with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency: facts and artefacts. The organic acid excretion in a patient with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency is described. In addition to 3-hydroxy-3-methylglutaric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxyisovaleric acids, substantial amounts of glutaric and adipic acids were repeatedly observed. 3-Methylcrotonic acid and possibly one of its isomers was found to be formed artificially from 3-methylglutaconic acid.


Effect of piperazine oestrone sulphate on serum oestrogen and gonadotrophin levels in post-menopausal women. Thirty-three post-menopausal women were treated with piperazine oestrone sulphate 1.5--3.0 mg daily on a cyclical basis for 3--6 months. Serum luteinising hormone (LH), follicle-stimulating hormone (FSH), oestrone and oestradiol were measured on two occassions prior to starting treatment, and several times during the course of therapy. Initial mean concentrations of oestrone and oestradiol of 59 and 14 pg/ml rose to 528 and 83 pg/ml, respectively, and this was associated with a dose-related suppression of LH (from 57.1 to 50.3 u/l) and more particularly FSH (from 25.8 to 16.5 u/l). There was a highly significant correlation between the decrease in FSH and the oestradiol concentration during treatment, but no correlation with oestrone.


The relationship between circadian variations in circulating thyrotrophin, thyroid hormones and prolactin. Half-hourly blood samples were taken from six clinically euthyroid men over a continuous period of 24 h. Their concentrations of total thyroxine (T4), total triiodothyronine (T3), thyrotrophin (TSH) and prolactin (PRL) were assessed together with the degree of unsaturation of thyroid hormone binding proteins as determined by the thyroid hormone uptake test (THUT). Both T3 and T4 were also measured in urine samples collected serially during the same 24 h period. Significant circadian changes in serum TSH, THUT, serum and urine T4 and serum PRL were demonstrated in all subjects. TSH showed a reciprocal pattern to serum T4, with higher levels during the evening and at night than the daytime. This TSH pattern did not coincide with PRL secretion. Further studies on the same subjects did not show any significant effect of posture, corticosteroid or T4 administration upon circadian changes in TSH. There appeared to be no consistent circadian changes in serum or urinary T3. It seems likely that the TSH circadian rhythm is centrally determined and that free T3 levels are maintained more or less constant by variation in peripheral conversion from T4.


Thyroid iodine concentration and total thyroid iodine in normal subjects and in endemic goitre subjects. Thyroid iodine concentration and total thyroid iodine were determined by an automated technique by means of the ceric arsenious acid reaction in 42 males and 13 females. The thyroids were taken from autopsy material in a region with a low dietary intake. The accuracy of the method to estimate iodine was checked by neutron activation analysis. There was an excellent correlation between the iodine contents of thyroid specimens, when measured by chemical or by activation analysis. The mean iodine concentration was 1.86 mg/g dry weight and was found to be lower than in non-goitrous regions. In goitres, the mean iodine concentration was significantly lower, and the mean total thyroid of normal weight. There was no significant difference in iodine concentration and total iodine between males and females. The thyroid iodine concentration did not depend upon age in adults. The data were log normally distributed. It was established that mean iodine concentrations under 2 mg/g dry weight or 0.45 mg/g fresh weight are to be regarded as an important index of iodine deficiency.


A sensitive bioassay of parathyroid hormone in plasma. Understanding of calcium metabolism in health and disease has been retarded by the lack of an adequately sensitive bioassay of parathyroid hormone. The problem of dissociation of bioactivity and immunoactivity, well recognized for other polypeptide hormones, is exaggerated in the case of parathyroid hormone by the disproportionately long half-time in the circulation of the immunoreactive fragments. A new method of assaying the biological activity of parathyroid hormone in plasma has been developed, based on the cytochemical methods which have yielded highly sensitive bioassays of other polypeptide hormones. It depends on the stimulation of glucose 6-phosphate dehydrogenase activity in the distal convoluted tubules of segments of guinea-pig kidney maintained in vitro, and measured by microdensitometry. The limit of sensitivity of the assay is 5 fh/ml (bPTH); the index of precision is 0.09 +/- 0.04 (mean +/- SEM; n = 11).


Preliminary studies with the sensitive cytochemical assay for parathyroid hormone. A modification of the cytochemical assay for parathyroid hormone (PTH) is described which permits the measurement of as little as 0.1 fg ml-1. Using this assay circulating concentrations in normal humans range between 1 and 30 pg ml-1 using a bovine PTH standard. Elevated values are found in hyperparathyroidism with reduced concentrations in hypoparathyroidism. Where detectable, PTH concentrations measured by radioimmunoassay are on average fourteen-fold higher than by cytochemical assay. No cross reactivity was found with AVP, calcitonin and 1,25 dihydroxy vitamin D in concentrations normally found in health or disease.


Thyroxine, triiodothyronine and thyrotrophin levels in meningococcal meningitis, typhoid fever and other febrile conditions. Thyroid status was estimated serially by measuring triiodothyronine (T3), thyroxine (T4) and thyrotrophin (hTSH) in 20 patients suffering from meningococcal meningitis, typhoid fever and other acute febrile illnesses. Significantly low T3 and only slightly increased T4 were observed in all the patients. hTSH was normal in all of these. A significant reciprocal relationship was found between the degree of fever and fall in T3 concentrations. T3 tended to rise in patients who recovered but in those who deteriorated or died, T3 remained persistently low.


The relation between plasma androstenedione and oestrone levels in untreated and corticosteroid-treated post-menopausal women. Plasma oestrone (E1) and androstenedione (A) were measured in 96 untreated and 18 corticosteroid-treated post-menopausal women. Levels of both hormones were consistently lower in the steroid-treated cases, presumably due to suppression of ACTH secretion. There was a clear relationship between A and E1 levels in both groups and when the data were pooled they formed a continuous series with a curvilinear relationship going through the origin. This relationship could be described by an equation based on Michaelis-Menten kinetics, suggesting that the rate of A to E1 conversion is an inverse function of the plasma androstenedione level. The main determinant of plasma E1 was plasma A; body weight and age were of subsidiary importance. Secondary oestrogen deficiency may be a factor in the genesis of steroid-induced osteoporosis in post-menopausal women and oestrogen therapy may be indicated in this group of patients.