Insulin's structural behavior and its relation to activity

This paper discusses the hypothesis that insulin undergoes a conformational change either before or during its binding to the receptor. The evidence for this is not conclusive but allows us to reconcile the following observations: (1) no chemical modification or deletion of invariant surface residues has abolished the hormone's activity—only reduced its potency. (2) Reduction in potency follows many modifications to different side chains, both variant and invariant. (3) There are insulins with perfectly preserved structure (by the criteria of aggregation, spectroscopy, and x-ray analysis) that have markedly reduced potency. (4) Insulins with disturbed structure still exhibit real, sometimes substantial activity.     

Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.     

The effect in vivo of alterations in gonadotropins and cyclic nucleotides on oocyte maturation in the hamster

The temporal relationship of changes in cAMP and cGMP to oocyte maturation was examined in proestrous hamsters (day 4). The first series of experiments showed, in normal cycling hamsters, an increase in cAMP and a decrease in cGMP at 1400 h shortly after the rise in LH with oocyte maturation beginning at 1800 h. When a second group of animals was injected with phenobarbital at 1200 h to block the LH surge, no significant change occurred in either cyclic nucleotide and oocyte maturation was prevented. In the second series of experiments single injections of either saline, hCG (30 IU), LH (10 micrograms) or FSH (10 micrograms) were given each to a group of animals at 0900 h on day 4. Animals were killed at five time intervals between 15 min and 3 h following the injection. LH and hCG stimulated a simultaneous increase in cAMP and decline in cGMP. The injection of FSH, however, did not cause an increase in cAMP but still produced a sharp decline in cGMP. Oocyte maturation occurred at 3 h in those animals injected with gonadotropins. Animals injected with saline showed neither cyclic nucleotide changes nor oocyte maturation. When cAMP and cGMP levels were expressed as a ratio (cAMP/cGMP) a significant increase occurred in the normal cycling animals and in those injected at 0900 h with gonadotropins. Phenobarbital and saline injected control animals showed no significant increase in the cAMP/cGMP ratio and no oocyte maturation. The results of these experiments and previous studies by this investigator indicate that cGMP may play an important role in oocyte maturation in the hamster prior to the LH surge. Since, in the presence of gonadotropins, the cAMP/cGMP ratio increases prior to oocyte maturation, it may be that the cyclic nucleotide ratio is also of importance in this process. Previous work by Hubbard and Terranova (1) has shown that guanosine 3':5' cyclic monophosphate (cGMP), can inhibit spontaneous maturation of hamster oocytes in vitro. This inhibitory action was dose dependent and overcome by LH. The cGMP-mediated inhibition occurred only in cumulus-enclosed oocytes, while adenosine 3':5' cyclic monophosphate (cAMP) inhibited spontaneous maturation in both cumulus-enclosed and denuded oocytes. The results of this study suggested that cGMP may play a role in inhibiting oocyte maturation prior to the LH surge. LH, the initiator of oocyte maturation, has also been shown in the intact proestrous rat and hamster to cause a decrease in cGMP at the same time that cAMP is rising (2,3).(ABSTRACT TRUNCATED AT 400 WORDS)     

The differentiation of glial cells and glia limitans in organ cultures of chick spinal cord

Summary Differentiation of glial cells and the glia limitans in organ cultures of chick spinal cord explanted at early neural tube stages, alone or with adjacent tissues, was studied by electron microscopy. Oligodendrocytes and astrocytes comparable to those seen in the chicken in vivo were observed, mainly in areas of good neuronal differentiation. A glia limitans with basal lamina, comparable to that in vivo, was found when spinal cord was bordered by normally adjacent tissues. When it was surrounded by vitelline membrane only, a characteristic limiting layer of glial processes, but no basal lamina, was seen. Contact with a filter membrane (Millipore) elicited excessive differentiation of glial filaments and modified cell fine structure; no glia limitans was formed. Supported by Grant 5 RO 1 NB 0637 from the United States Public Health Service.     

Growth and lipolysis of rat adipose tissue: effect of age, body weight, and food intake

The purpose of the present work was to study age- and weight-controlled rats to determine which is the primary factor in reducing the lipolytic response of free fat cells and which has the greater effect on the ratio of fat cells to nonfat cells in adipose tissue. The method for estimating fat cell and nonfat cell numbers is based on the analysis of adipose tissue and fat cell DNA and lipid. In adequately fed rats, epididymal adipocyte hyperplasia is complete between 9 and 14 wk of age. Chronic underfeeding delays, but does not eliminate, normal fat cell hyperplasia and is accompanied by a net loss in the nonfat cell population. During 9-14 wk of age, rat epididymal adipose tissue enlarges mainly through adipocyte hypertrophy. Total fat cells from the epididymal adipose tissue of control rats represent only 20-23% of the total cell population. Chronic underfeeding increases the percentage of fat cells in the fat pad from 23 to 28%. Noradrenaline-stimulated lipolysis is proportional to fat cell numbers but is inhibited when fat cell lipid increases to over 80% of fat pad wet weight. Rat age is apparently not primarily responsible for the decreased noradrenaline-stimulated lipolysis in fat cells of 350-g rats in vitro.     

Time of origin of Mauthner's neuron in Xenopus laevis embryos

The time of the last DNA replication of the Mauthner's neuron precursor cell has been investigated using radioautography. Embryos of Xenopus laevis were labeled at different stages of early development by single microinjections of tritiated thymidine. Labeling times were designed to cover the entire period of development between gastrula and hatching stages. The embryos were fixed at later stages (41 to 44, according to Nieuwkoop and Faber, 1967), when the Mauthner neuron can be readily distinguished by its characteristically large size and large nucleolus.Mauthner neurons of embryos which received tritiated thymidine from stage 10 (beginning of gastrulation) to stage 12 (advanced gastrula, medium yolk plug) were always labeled. Those embryos which received the isotope at or after stage $\text{12}\text{1}\text{2}$ (advanced gastrula, small yolk plug) were never found labeled. These results imply that the last DNA replication of the cell destined to give rise to the Mauthner neuron occurs during the last gastrula stages. This last DNA replication immediately proceeds the time of the so-called “histogenetic determination” of the Mauthner neuron proposed to correspond to stage 13 (slit blastopore) by Stefanelli (1951).Therefore it appears that the developmental program of the Mauthner neuron involves a remarkably early cessation of DNA replication closely followed by histogenetic determination. This is the earliest known event of this type for a specific, well characterized neuron in the amphibian embryo.     

Applicability of radiotracer methods of measuring14CO2 assimilation for determining microbial activity in soil including a newin situ method

The applicability of two methods (pyrolysis gas chromatography and acidification-wet oxidation) for determining¹⁴CO₂ incorporation into soil microorganisms was investigated. Both methods were able to distinguish biologically incorporated¹⁴C from abiotically adsorbed¹⁴C, but to varying degrees, there being a larger carryover of abiotic¹⁴C into the organic fraction and a higher percentage of assimilated¹⁴C in the organic fraction with the wet oxidation method. Using¹⁴C assimilation as a measure, it was possible to determine microbial activities in soils of diverse properties under a variety of conditions, including polar soils under harsh environmental conditions. Both light and dark¹⁴CO₂ fixation was measurable.¹⁴CO₂ assimilation was not always proportional to the enumerable microorganisms. A new design for measurement of microbial activityin situ enabled measurement of total C influx (primary productivity) into soils with minimal perturbation to the natural soil ecosystem.