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Biomedical subjects

M W Berchtold

Publications and source records attributed to M W Berchtold.

67 records · Page 4Linked to original sources
PubMed1987

Changes in the concentration of the calcium-binding parvalbumin in cross-reinnervated rat muscles. Comparison of biochemical with physiological and histochemical parameters.

The fast extensor digitorum longus (EDL) and the slow soleus (SOL) muscles were cross-reinnervated in both directions in the rat. During the following transformation of muscle type properties, the expression of the Ca2+-binding parvalbumin (parvalbumin, Mr = 12,000) was investigated. The combined biochemical, histochemical, and physiological results demonstrated that the amount of parvalbumin decreased in the fast to slow (X-EDL) and increased in the slow to fast (X-SOL) transformation. Alterations of parvalbumin-mRNA levels were similar to changes found at the protein level, indicating a tight transcriptional regulation of the parvalbumin expression. The close correlation, however, between parvalbumin and relaxation speed found in normal muscles had changed after cross-reinnervation. After the altered nervous input, a slow contracting/slow relaxing muscle may even contain more parvalbumin than a fast contracting/fast relaxing one. The expression of parvalbumin may depend on the nerve-muscle interaction, and parvalbumin may thus be used as a sensitive marker for early stages of muscular transformation and neurological disorders.

Animals↗
PubMed1986

Isolation of a rat parvalbumin gene and full length cDNA.

The complete sequence of the rat parvalbumin mRNA was determined by sequencing a near full length cDNA clone and a primer extension product of the 5' untranslated region of this clone. The parvalbumin sequence contained 72 nucleotides of the 5' untranslated region, 333 nucleotides of the coding sequence, and 183 nucleotides of the 3' untranslated sequence in the case of the shorter parvalbumin messenger RNA or 556 nucleotides in the case of the larger parvalbumin messenger RNA. The two RNAs were derived from a single primary transcript since two clones were sequenced which were identical except for the site of polyadenylation. The polyadenylation signal of the shorter, more abundant RNA was AATAAA while the putative signal for the larger RNA was GATAAA. A 32-base pair sequence comprising part of the 5' untranslated region and the first seven codons of the parvalbumin cDNA was 81% homologous to a region of the chicken calmodulin cDNA encoding part of the first Ca2+-binding domain. This homology indicates that this portion of the parvalbumin mRNA may have evolved from a primordial first domain. A parvalbumin genomic clone which hybridized to probes derived from both the coding and 3' untranslated region of the cDNA was cloned from a rat genomic library. Partial sequencing identified an uninterrupted stretch of 585 base pairs identical to the 3' end of the parvalbumin cDNA. Two introns were localized in the genomic clone. Their position with respect to the parvalbumin amino acid sequence corresponded to the intron locations found in genes of several other Ca2+-binding proteins. This result indicates that, despite the deletion of one Ca2+-binding domain, the remainder of the parvalbumin gene has maintained the structural pattern of the Ca2+-binding protein gene family.

Amino Acid Sequence↗
PubMed1986

Skin calcium-binding protein is a parvalbumin of the panniculus carnosus.

Skin calcium-binding protein (SCaBP) is a calcium binding protein purified from whole rat skin. It has a molecular weight of approximately 12,000 daltons but migrates at Mr 13,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gels. On nitrocellulose blots of SDS-polyacrylamide gels, 6 different antisera to SCaBP reacted equally well with SCaBP and parvalbumin (PV), an 11,500-dalton calcium-binding protein purified from rat skeletal muscle, which also migrates at Mr 13,000 on SDS-polyacrylamide gels. Rabbit antiserum to muscle PV also recognized both PV and SCaBP, and either protein absorbed specific antibodies against either antigen from both types of antisera. Soluble protein extracts from whole adult rat and mouse skin contained a Mr 13,000 protein which was recognized on nitrocellulose blots of SDS gels by both antisera. Blots of extracts from epidermis, dermis, whole skin, and skin scraped on the dermal side to remove hypodermal tissue revealed that the Mr 13,000 PV/SCaBP cross-reacting antigen was restricted to the hypodermal tissue removed by scraping. Immunofluorescent staining of Bouin-fixed skin sections with these antisera confirmed the localization of PV/SCaBP to the panniculus carnosus, a hypodermal muscle layer. Newborn mouse skin does not contain this antigen. Additional polypeptides of Mr 10,500 and 12,000 on SDS gels of extracts from the epidermis of newborn and adult rats and mice were found to be immunoreactive with anti-SCaBP serum. These polypeptides were not recognized by the PV antiserum, and the reactivity of anti-SCaBP for these antigens was not absorbed by purified PV or SCaBP. Our results indicate that SCaBP is antigenically indistinguishable from PV and is localized in the adult rodent panniculus carnosus, and that antisera to SCaBP are polyspecific, recognizing epidermal proteins in addition to SCaBP/PV.

Animals↗
PubMed1985

Parvalbumin in cross-reinnervated and denervated muscles.

The extensor digitorum longus (EDL) muscle was cross-reinnervated by the soleus (SOL) nerve, leading to the well-known transformation toward a slow muscle. Nine weeks after the operation, the quantitative analysis of the Ca2+-binding protein, parvalbumin (PV), using high-performance liquid chromatography, showed a threefold reduction of PV in the cross-reinnervated EDL muscle. Denervation of the EDL muscle, which leads to an increase of the half-relaxation time, resulted in a 20% decrease of the PV concentration within 4 days. This significant lower PV level was detectable prior to any change of the myofibrillar adenosine triphosphatase (ATPase). Normal PV concentrations were reached after 9 weeks following self-reinnervation of the EDL muscle. The experiments support the view that PV is involved in the relaxation of rat fast skeletal muscles and that its expression is dependent on nerve-muscle interaction. Since PV changes preceded histochemical changes after denervation, this protein may be a sensitive marker for early stages of neuromuscular disturbances.

Adenosine Triphosphatases↗
PubMed1985

The Ca2+-binding protein parvalbumin: molecular cloning and developmental regulation of mRNA abundance.

Parvalbumin (PV) is a Ca2+-binding protein found only in vertebrates. It is postulated to serve as a soluble relaxing factor in fast mammalian muscle. We have isolated a rat PV cDNA clone and used this as a probe to examine changes in PV mRNA during muscle and brain development. A cDNA library was constructed in PUC8/PUC9 plasmid vectors from adult poly(A)+ RNA isolated from rat gastrocnemius muscle. The library was screened with a 17-mer oligonucleotide encoding amino acids 28-33 of rat PV. One recombinant (9f) was confirmed as a PV clone by DNA sequencing and was shown to contain 73% of the protein coding sequence. Hybridization of clone 9f to RNA separated by electrophoresis revealed two species 700 and 1100 nucleotides long but genomic blotting indicates that PV may be a single copy gene. Highest levels of PV mRNA are found in the gastrocnemius, which is a fast contracting/relaxing muscle. Skin contains the next highest amount of PV mRNA followed, in order, by brain and the slow twitch soleus muscle. Rat muscle PV mRNA levels increase 15- to 20-fold between postnatal days 4 and 20 as measured by dot blot hybridization of total RNA, whereas only a slight increase was observed when young and adult brains were compared. The changes in PV mRNA during development appear to be selective, because mRNA coding for the structurally homologous Ca2+-binding protein calmodulin (CaM) was found to change only slightly in muscle. However, CaM mRNA levels decrease during the early days of brain ontogeny. Thus, the mRNAs that encode the homologous Ca2+-binding proteins PV and CaM appear to be developmentally regulated in a tissue-specific manner.

Age Factors↗
PubMed1985

Parvalbumin in human brain.

Parvalbumin was isolated from human cerebral cortex and biceps and triceps muscles by HPLC. The immunological properties of the human protein and the mobility in two-dimensional polyacrylamide gels were similar to that of parvalbumin isolated from the muscles of rat, mouse, rabbit, and chicken. The tryptic peptide maps of the human parvalbumin, however, differed considerably from all other parvalbumins, indicating a distinct primary structure. The immunolabeled cells in the hippocampus of the human brain were of different sizes and forms; they occurred in all subfields and probably represent interneurons.

Adult↗
PubMed1984

Parvalbumin in non-muscle tissues of the rat. Quantitation and immunohistochemical localization.

Parvalbumin, a high affinity Ca2+-binding protein, is known to be expressed only in muscles and brain in the rat. We have investigated its distribution and characteristics in other rat tissues by several biochemical and immunohistochemical methods. Evidence for the presence of parvalbumin in teeth, bone, skin, prostate, seminal vesicles, testes, and ovary is given by two-dimensional polyacrylamide gel electrophoresis, immunoblotting ("Western technique") of one-dimensional gels, and its concentration measured by reverse phase high performance liquid chromatography. The distribution within several parvalbumin-positive organs was monitored by the immunohistochemical peroxidase-antiperoxidase method. In teeth, only ameloblasts reacted with anti-rat parvalbumin serum and in bone the calcified extracellular cartilage was the target of the immunoreaction. The panniculus carnosus was the exclusive site of parvalbumin in the skin. Besides the already known parvalbumin distribution in the brain, parvalbumin is also expressed in distinct cell types of the peripheral nervous system. Leydig cells were found to be the only parvalbumin location in testes. These observations lead us to conclude that parvalbumin in contrast to the multifunctional and constitutive calmodulin must function in Ca2+-dependent processes related to specific cell types.

Animals↗
PubMed1983

Ca2+-binding proteins: a comparative study of their behavior during high-performance liquid chromatography using gradient elution on reverse-phase supports.

Reverse-phase high-performance liquid chromatography has been shown to be applicable to the isolation of Ca2+-binding proteins, specifically parvalbumins, from tissue extracts or from preparations first purified by "conventional" chromatography. Through an investigation of the behavior of a series of Ca2+-binding proteins as a function of buffer composition, pH, and organic eluant it has been possible to define mild conditions allowing for chromatography of the proteins in their native states. The elution positions of parvalbumins were not observed to correlate with the "overall" protein hydrophobicity, calculated using hydrophobicity values for the individual amino acids, thus indicating that factors such as hydrophobic/hydrophilic surface areas are important in determining the degree of association with the support. The usefulness of reverse-phase chromatography as an analytical tool for determining protein homogeneity is illustrated. Samples which had been isolated via "conventional" chromatography methods, and thought to be homogeneous, were observed to contain multiple species of the same protein.

Animals↗
PubMed1982

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin.

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.

Animals↗
PubMed1982

Correlation of parvalbumin concentration with relaxation speed in mammalian muscles.

The physiological role of the Ca2+-binding protein parvalbumin in skeletal muscle has been investigated by measuring the parvalbumin content by HPLC in a variety of mammalian muscles, including man, and comparing the results with the respective muscle relaxation properties and fiber type compositions. The parvalbumin concentrations were highest in the skeletal muscles of the smallest animal investigated (mouse, gastrocnemius: 4.9 g/kg), which has the highest relaxation speed, and lowest in the larger animals (horse, deep gluteal muscle: less than or equal to 0.001 g/kg) and man (vastus, triceps: less than or equal to 0.001 g/kg), which have much lower relaxation speeds. Analysis of three type-homogeneous muscles of the guinea pig revealed highest parvalbumin concentrations (0.25 g/kg) in sartorius (type IIB) and lowest concentrations (less than or equal to 0.007 g/kg) in soleus (type I), consistent with the different half-relaxation times of fast and slow muscles. Denervation of the rat extensor digitorum longus, which increases the half-relaxation time from 9.4 to 19 msec, resulted in a 20% decrease of the parvalbumin content. Given this close correlation between parvalbumin content and relaxation speed in a variety of muscles and species, we suggest that parvalbumin is involved directly in the relaxation process in fast muscles.

Animals↗
PubMed1982

Primary structure of parvalbumin from rat skeletal muscle.

The primary structure of parvalbumin from rat skeletal muscle has been determined principally by automated sequencing of tryptic peptides using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate as the Edman reagent on a solid-phase sequencer. Remaining positions and most peptide overlaps were identified by analysis of peptides arising from CNBr, chymotryptic and Staphylococcus aureus protease cleavages and through digestions with carboxypeptidases A, B and Y. Reverse-phase high-performance liquid chromatography on C-18 supports was employed for all peptide separations. Structural homology between rat and rabbit parvalbumins helped to confirm the alignments of the tryptic peptides T4-T3, T2-T6 and to define the position of the Lys triplet (36-38). A comparison of the two mammalian proteins revealed 14 amino acid differences, which are all located on the surface of the molecule. A prediction of the secondary structure has been made and found to be very similar for the rat and rabbit proteins with the exception of the sequence region 72-78, located between the Ca2+, Mg2+-binding CD and EF domains.

Amino Acid Sequence↗