The α6 integrin subunit in the developing mouse olfactory bulb

Integrins are heterodimeric cell surface receptors that mediate developmental events by binding extracellular matrix ligands. Several lines of evidence suggest a role for integrins, specifically the α 6 subunit, in neuronal migration, neurite outgrowth, and axon guidance during olfactory development. Therefore, we undertook an analysis of the expression of the α 6 subunit in the olfactory system of the embryonic and early postnatal mouse to understand the role it may play during neural development. In addition, as a functional assay we examined the developmental effects of the loss of this subunit on olfactory development by analyzing an α 6 knockout (α 6^?/?). Immunohistochemical analyses and confocal microscopy were used to examine α 6 expression in the CD-1 embryonic and early postnatal olfactory system and also to examine the organization of the olfactory system in the α 6^?/? mouse. In CD-1 mice from E13 to E17, α 6 localizes in radial patterns extending from the core of the olfactory bulb to the nerve layer and colocalizes with RC2, an antibody specific for radial glia. By the day of birth (P0; ～E19), expression is limited to the external plexiform layer and the olfactory nerve layer, where it colocalizes with laminin and p75. In the α 6^?/? mouse, areas of ectopic granule cells were observed in the mitral cell layer of the olfactory bulb. These ectopias coincided with areas of disorganization of the radial glial processes and breaks in the mitral cell layer. These observations suggest a role for α 6 integrin in neural migration during olfactory development, likely secondary to organization of the radial glial scaffold.     

Neuropeptides in the pineal gland?

Summary In an attempt to localize components of the renin angiotensinsystem in the pineal gland of rats, immunocytochemical studies using the PAP-technique were performed with antisera against angiotensin I, angiotensin II and angiotensinogen. The staining pattern thus obtained was not only the same for the three antisera, but was also identical to that shown for many other peptide-antisera in the literature. In those studies, the immunocytochemical staining had been ascribed to a distinct pineal cell population or to cell processes. However, by examining adjacent semithin and ultrathin sections by immunocytochemistry and electron microscopy, respectively, we could identify the extracellular perivascular compartment and its flocculent material as the site of staining.This unexpected localization and the observation of immunoreactivity of some preimmunsera in the same compartment as well as several additional findings and arguments are taken to suggest the likelihood of pseudopositive immunostaining, typical for the pineal gland.These studies were supported by the German Research Foundation within the SFB 90 Cardivasculäres System     

The differential actions of cortisol on the accumulation of α-lactalbumin and casein in midpregnant mouse mammary gland in culture

The dose-response relationship between cortisol and the accumulation of the two milk proteins, casein and α-lactalbumin, was studied in organ culture of mammary gland from midpregnant mice. The accumulation of casein was low in culture with insulin but was enhanced by the further addition of prolactin. Further increases in casein were effected by the addition of cortisol in increasing concentrations up to 3 × 10^?6 M, which was optimal for the accumulation of this protein. The content of α-lactalbumin in explants was similarly low in culture with insulin alone, but, in contrast, was increased to a maximal level by the addition of insulin and prolactin. The addition of cortisol up to 3 × 10^?8 M with insulin and prolactin did not further increase the level of α-lactalbumin; in fact, at concentrations above 3 × 10^?7 M the steroid caused progressive inhibition of the accumulation of this protein in cultured explants. Studies of the appearance of casein and α-lactalbumin in incubation medium during organ culture revealed the presence of substantial amounts of these milk proteins. During the first 2 days of culture with insulin, prolactin and 3 × 10^?6 M cortisol, the amount of α-lactalbumin in culture medium was almost equal to the level found in tissue, whereas in the presence of 3 × 10^?8 M cortisol, or in the absence of exogenous steroid, over 70% of total α-lactalbumin was retained in tissue. The observed difference in the amount of α-lactalbumin in culture medium can, however, only partially account for the inhibitory effect of high doses of cortisol on the accumulation of α-lactalbumin in cultured mammary explants. In contrast to α-lactalbumin, the relative amount of casein in culture medium containing insulin and prolactin was smaller—19% of total casein synthesized—and was further reduced to 16% and 11% of the total in the presence of 3 × 10^?8 M and 3 × 10^?6 M cortisol, respectively. The above results indicate that cortisol exerts dose-dependent differential actions on the accumulation of casein and α-lactalbumin in mouse mammary epithelium in vitro.     

Nyctohemeral rhythmicity of type II thyroxine 5′-deiodinase activity in the pineal gland but not in the Harderian gland of the Swiss mouse

Type II thyroxine 5-deiodinase (5-D) activity in both pineal and Harderian glands of the Swiss mouse was studied. Pineal 5-D activity exhibited a nyctohemeral profile with a maximal peak value at 05.00 h, which coincides with that for pineal melatonin production. However, no rhythm of 5-D activity in the Harderian gland could be found. In pineal gland, light at night inhibited the nocturnal increase in 5-D activity, while isoproterenol, a -adrenergic agonist, could not stimulate the enzyme. In the Harderian gland, neither darkness, nor light at night, or isoproterenol were capable of modifying basal values of 5-D activity.     

Colocalization of pinopsin with two types of G-protein α-subunits in the chicken pineal gland

Pinopsin is a photoreceptive molecule present in the outer segments of chicken pinealocytes. In this paper, the localization of alpha-subunits of G-proteins, rod transducin (Gt1) and Gq/11, was examined by immunoelectron microscopy to investigate whether these G-proteins colocalize with pinopsin in the outer segments. Ultrathin sections of the chicken pineal gland were double-immunolabeled with antibodies to pinopsin and either Gt1alpha or Gq/11alpha. As shown previously, the outer segments around the follicular lumen exhibited divergent morphology with ciliary, bulbous, or lamellate shapes, and most of them displayed pinopsin immunoreactivity. The majority (>90%) of pinopsin-immunopositive outer segments were labeled by anti-Gt1alpha and/or anti-Gq/11alpha antibodies. Application of double-immunolabeling to serial sections demonstrated that a large number of the pinopsin-immunopositive outer segments contained both Gt1alpha and Gq/11alpha immunoreactivities. These results suggest that Gt1alpha and Gq/11alpha are functionally coupled with light-activated pinopsin within a single outer segment.     

Protein kinase C isoenzymes in mouse Harderian gland. Differential expression of the α- and ε-isoforms during pregnancy

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-,-,- and-. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the - and -isoforms. The 80-kDa native form of PKC- almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C- appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant inthe Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions. Moreover, the expression of the - and -isoforms could be regulated by sexual hormones, as suggested by the differential abundance of these two proteins in the gland of pregnant mouse compared with normal female mouse.     

Regional expression and subcellular localization of the voltage-gated calcium channel β subunits in the developing mouse brain

J. Neurochem. (2012) 122, 1095-1107. ABSTRACT: Ca(2+) channel β subunits determine the maturation, biophysical properties and cell surface expression of high voltage-activated channels. Thus, we have analysed the expression, regional distribution and subcellular localization of the Ca(v) β subunit family in mice from birth to adulthood. In the hippocampus and cerebellum, Ca(v) β(1) , Ca(v) β(3) and Ca(v) β(4) protein levels increased with age, although there were marked region- and developmental stage-specific differences in their expression. Ca(v) β(1) was predominantly expressed in the strata oriens and radiatum of the hippocampus, and only weakly in the cerebellum. The Ca(v) β(3) subunit was mainly expressed in the strata radiatum and lucidum of the hippocampus and in the molecular layer of the cerebellum. During development, Ca(v) β(3) protein expression in the cerebellum peaked at postnatal days (P) 15 and 21, and had diminished drastically by P60, and in the hippocampus increased with age throughout all subfields. Ca(v) β(4) protein was detected throughout the cerebellum, particularly in the molecular layer, and in contrast to the other subunits, Ca(v) β(4) was mainly detected in the molecular layer and the hilus of the hippocampus. At the subcellular level, Ca(v) β(1) and Ca(v) β(3) were predominantly located post-synaptically in hippocampal pyramidal cells and cerebellar Purkinje cells. Ca(v) β(4) subunits were detected in the pre-synaptic and post-synaptic compartments of both regions, albeit more strongly at post-synaptic sites. These results shed new light on the developmental regulation and subcellular localization of Ca(v) β subunits, and their possible role in pre- and post-synaptic transmission.     

Presence of AVT-, α-MSH-, LHRH- and somatostatin-like compounds in the rat pineal gland and their relationship with the UMO5R pineal fraction

Using antibodies against AVT, alpha-MSH, LHRH and somatostatin, immunoreactive cells were detected in the rat pineal gland. All of these antibodies stain the same cells, which also react immunocytochemically when an antibody against the UMO5R sheep pineal fraction, a fraction that presents antigonadotropic properties in vivo, is used. Relatively more immunoreactive cells are present in the pineals of young rats than in the pineals of adult animals. Comparison of the results obtained with different potent antibodies against each of the peptides, and a study of the staining properties of the antibodies in the pineal after solid phase absorption to different peptides or to different sheep pineal fractions, led to the proposal that the immunoreactivity found in the rat pineal is not due to the presence of AVT, alpha-MSH, LHRH or somatostatin, but to a cross-reaction of each of these antibodies with (an) unidentified compound(s). This compound is synthetized in the pineal gland, as was demonstrated using cultured pineals. The UMO5R and the Prot. 4 fractions of the sheep pineal seem to be chemically related to this unknown compound, the possible endocrine nature of which is discussed.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of thehα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

a-lactalbumin(a-Lac),amajorwheyprotein,isacalciummetalloprotein,thathasbeenfoundinallmilksstudiedsofar.ItinteractswithUDP-galactosyl-transferasetoformthelactosesynthetaseandthusmightbeakeyproteinforlactogenesis.Lactosesyn-thetaseispostulatedtobetherate-limitingenzymeforlactosebiosynthesis.Theincreaseda-Lacactivitycanproducesufficientlactosesynthetaseforthesynthesisoflactose,andinmilkyieldbydrawingwaterintomilk,sincelactoseisanosmoreactivemolecule.Transgenicswineoverexpressingbovinea-lactalbu…     

Rat α2u-globulin mRNA expression in the preputial gland

Rat _2u-globulin and the mouse major urinary proteins (MUP) are encoded by homologous multigene families whose members exhibit diverse tissue-specific, developmental, and hormonal controls of expression. Although their patterns of expression and hormonal control appear to be very similar in many respects, we have found high levels of _2u-globulin mRNA in rat preputial glands, whereas MUP mRNA could not be detected in the male mouse preputial gland. Male and female rat preputial have similar concentrations of _2u-globulin mRNA, suggesting an absence of endocrine regulation as occurs in the liver and lachrymal glands. Two-dimensional polyacrylamide gel electrophoresis of proteins encoded by hybrid-selected _2u-globulin mRNA indicates that the liver and lachrymal translation products have different mobilities. However, many of the preputial gland products comigrate with most or all of the liver and lachrymal products. Among the possibilities suggested by these results is that _2u-globulin genes expressed in liver and lachrymal glands under endocrine control are also expressed constitutively in the preputial gland.This work was supported by Public Health Service Research Grant GM25023 from the National Institutes of Health.     

Studies on the particulate lactose synthetase of mouse mammary gland and the role of α-lactalbumin in the initiation of lactose synthesis

1. Some of the kinetic properties of the particulate lactose synthetase of mouse mammary gland were investigated and shown to resemble those that have been reported for the soluble enzyme. Typical values for intact preparations were 2.3mum for the apparent K(m) for alpha-lactalbumin at 40mm-glucose and 1.7mm for the apparent K(m) for glucose at the endogenous concentration of alpha-lactalbumin. 2. Digitonin treatment increased total assayable activity approximately twofold but almost eliminated the endogenous activity found in the absence of alpha-lactalbumin, these findings being consistent with the location of endogenous activity within Golgi vesicles. 3. From the properties of the particulate fraction from lactating mice it was deduced that the effective endogenous alpha-lactalbumin concentrations were in the range 1-10mum. 4. The concentration of alpha-lactalbumin was not significantly different in particles isolated at various stages of pregnancy and early lactation. 5. The implications of these results for the control of lactose synthetase in vivo are discussed.     

How the projection domains of NF-L and α-internexin determine the conformations of NF-M and NF-H in neurofilaments

Making use of a numerical self-consistent field method and polymer brush concepts, we model the solvated corona of neurofilaments (NF) composed of projection domains (unstructured tails) of constituent proteins. Projections are modeled with amino acid resolution. We focus on the importance of the two shortest ones (α-internexin and NF-L) in regulating the conformations of the two longer ones (NF-M and NF-H) in an isolated NF. We take the wild-type NF with no α-internexin as the reference, for which the phosphorylation-induced translocation of M- and H-tails has been examined previously. We demonstrate that a subbrush of L-tails creates an electrostatic potential profile with an approximately parabolic shape. An experimentally relevant (2:1) ratio of L- to α-projections reduces the charge density of the L subbrush and shifts the translocation transition of the H-tails to slightly higher degrees of phosphorylation. Replacing all L-tails by α-projections destroys the substructure of the NF corona and this alters the NF response to the phosphorylation of long tails.     

Tissue-specific expression of mouse α-amylase genes: Nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland

We have determined the nucleotide sequence of two different mouse α-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively, are the major α-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5′ noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict α-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland α-amylase mRNA is not initiated at the AUG codon nearest the 5′ terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.     

Expression and function of heregulin-α and its receptors in the mouse mammary gland

Heregulin-α (HRGα) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand the role of HRGα and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine the expression and localization of HRGα and its receptors ErbB2 and ErbB3. We also determined the role of HRGα in the development of mammary glands, β-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGα signaling molecules using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated with HRGα, indicating that HRGα positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGα enhanced the phosphorylation of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial cells of mammary glands, and increased and maintained the expression and secretion of β-casein. During lactation, HRGα enhanced the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology of the mammary glands and increased the secretion of lactoprotein to reduce the expression of β-casein in mammary epithelial cells. During involution, HRGα induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of β-casein, resulting in increased levels of β-casein in mammary epithelial cells.     

Cellular aspects of aging in the pineal gland of the shrew, Crocidura russula

The Greater White-toothed shrew Crocidura russula is short-lived species and the phase of senescence is greatly elongated in captivity. The loss of rhythmicity of biological functions that accompanies its aging is also well documented. C. russula is thus an excellent model to test the effects of aging on biological clocks. Melatonin is a key hormone in the synchronization of behaviors, metabolisms and physiological regulations with environmental factors. In the present work we want to know if the loss of rhythmicity and the reduced melatonin levels registered by the second year of life in this species could be associated to modified ultrastructural features of the pineal parenchyma, site of melatonin synthesis. Transmission electron microscopy (TEM) analysis of young (1-4 months) and old (25-28 months) shrew's pineals show that in older individuals, the parenchyma undergoes alterations affecting mainly nucleus, mitochondria and endoplasmic reticulum cisternae, with increased numbers of dense bodies and the formation of many concretions as well as a depletion of secretory products. These changes suggest a process of slowing pinealocytes metabolism which could explain the gradual reduction of melatonin levels registered during aging in C. russula.