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Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimer's disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine β-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.

We describe methods for the preservation, extraction and amplification of DNA from faeces that facilitate field applications of faecal DNA technology. Mitochondrial, protein encoding and microsatellite nuclear DNA extracted and amplified from faeces of Malayan sun bears and North American black bears is shown to be identical to that extracted and amplified from the same individual's tissue or blood. A simple drying agent, silica beads, is shown to be a particularly effective preservative, allowing easy and safe transport of samples from the field. Methods are also developed to eliminate the risk of faecal DNA contamination from hair present in faeces.

The gene for the iron-binding protein transferrin is transcribed at a high level in liver hepatocytes but is also active in several other cell types, including oligodendrocytes in the brain. Enhancer elements between bp -560 and -44 of the transferrin gene promoter specifically activated transcription from a heterologous promoter in transgenic mouse liver and brain. Within this region, a potent cis-acting element between bp -98 and -83 was found to be essential for gene activity in both cultured hepatocytes and transgenic mouse liver. The -98 to -83 element contains a CCAAT sequence and is specifically bound by a nuclear factor from mouse liver that is homologous to rat liver C/EBP (CAAT enhancer-binding protein). Point mutations within this binding site inhibit factor binding and abolish transcription in transfected hepatoma cells. When placed in the context of the 3,000-bp transferrin promoter, the C/EBP binding site mutation causes a complete loss of transcription in transgenic mouse liver; however, transgene expression in the brain of the same animals was unaffected. These results suggest a modular structure for the transferrin promoter and demonstrate that deletions or specific point mutations can be used to generate transgene promoters with an activity more restricted than that of their endogenous counterparts.

Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties. We have used recombinant techniques to express and purify the type I regulatory subunits, RI alpha and RI beta, the catalytic subunits C alpha and C beta, and then reconstituted holoenzymes with the various combinations of R and C subunits. The ability of the subunits to form inactive holoenzymes and then to be activated in the presence of cyclic nucleotides was examined. Holoenzymes containing C beta had essentially the same activation properties exhibited by C alpha holoenzymes. However, the presence of the neural form of RI, RI beta, led to formation of a holoenzyme which was activated at a 3-7-fold lower concentration of cyclic nucleotides compared to holoenzymes containing RI alpha. Expression of the RI beta protein in discrete regions of the central nervous system may provide a mechanism for increasing the sensitivity of the kinase to what would otherwise be subthreshold levels of stimulation. Two mutant forms of RI beta were constructed that converted the RI beta sequence to that of RI alpha at position 98 (RI beta Ala) or positions 98 and 99 (RI beta Ala/Ile). These sequences form part of a pseudosubstrate site thought to interact with the C subunit. Wild type and mutant R subunits were combined in vitro with purified bovine C subunits and half maximal activation constants (Ka) were determined with cyclic nucleotides. Holoenzymes containing RI beta Ala and RI beta Ala/Ile gave Ka values which were higher than wild type RI beta, with the double mutant shifting toward the Ka value of RI alpha holoenzymes by about 30%. These results suggest that amino acid differences in the pseudosubstrate site may account for some, but not all, of the increased sensitivity to cyclic nucleotides exhibited by RI beta.

Cyclic AMP (cAMP) and cAMP-dependent protein kinases (PKAs) are believed to be involved in the regulation of essential spermatozoal functions, such as motility, epididymal maturation, capacitation, and the acrosome reaction. In this study, we document the presence of significant mRNA levels for 5 different PKA subunits (RI alpha, RI beta, RII alpha, RII beta, and C alpha) in germ cells and demonstrate differential expression patterns for these subunits during spermatogenesis. Messenger RNAs for RI (RI alpha and RI beta) and C alpha appear to be induced at premeiotic germ cell stages, whereas mRNAs for RII (RII alpha and RII beta) are first expressed at haploid stages. The individual PKA subunits may convey specific functions in developing germ cells and mature sperm. The present study, furthermore, demonstrates the presence of unique smaller-sized mRNAs in germ cells compared with somatic cells. Specific, truncated forms of RI alpha, RII alpha, RII beta, and C alpha mRNAs appear to be selected in the germ cells. Our data suggest this to be due to the use of alternative polyadenylation site signals. The selection of shorter mRNA species, with higher stability, may be essential for the delayed translation observed in spermatids. This may ensure certain levels of mRNA for translation at late spermatid stages, after cessation of transcription.

Male Japanese quail chicks of two genetic lines selected for low (LS) or high (HS) adrenocortical responses to mechanical restraint were housed in mixed-line groups of 24 in four compartments of a multitier brooder battery at 20 days of age. Quail in two of the four compartments were given vitamin C (ascorbyl-2-polyphosphate, APP, 1 g L-ascorbic acid/L) solution for 48 h, whereas the other birds received untreated tap water as usual before they were tested at 23 days of age. At test, each quail was placed individually in a dark, sheltered compartment of an emergence box and allowed 1 min to acclimatise before a door was raised allowing access to an illuminated and exposed area. Vocalisation and the latencies to head and full emergence were then recorded to measure its fear levels. More LS quail vocalised than did HS ones. They also emerged more rapidly from the sheltered compartment into the illuminated one than HS birds. These findings further support our hypothesis that decreased fearfulness has accompanied genetic selection for reduced adrenocortical responsiveness. Treatment with APP reduced the latency to emerge fully into the exposed compartment, and there were no line x treatment interactions. These results suggest that vitamin C supplementation alleviated fearfulness, regardless of existing line differences in this behavioural trait.

Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties. We have used recombinant techniques to express and purify the type I regulatory subunits, RI alpha and RI beta, the catalytic subunits C alpha and C beta, and then reconstituted holoenzymes with the various combinations of R and C subunits. The ability of the subunits to form inactive holoenzymes and then to be activated in the presence of cyclic nucleotides was examined. Holoenzymes containing C beta had essentially the same activation properties exhibited by C alpha holoenzymes. However, the presence of the neural form of RI, RI beta, led to formation of a holoenzyme which was activated at a 3-7-fold lower concentration of cyclic nucleotides compared to holoenzymes containing RI alpha. Expression of the RI beta protein in discrete regions of the central nervous system may provide a mechanism for increasing the sensitivity of the kinase to what would otherwise be subthreshold levels of stimulation. Two mutant forms of RI beta were constructed that converted the RI beta sequence to that of RI alpha at position 98 (RI beta Ala) or positions 98 and 99 (RI beta Ala/Ile). These sequences form part of a pseudosubstrate site thought to interact with the C subunit. Wild type and mutant R subunits were combined in vitro with purified bovine C subunits and half maximal activation constants (Ka) were determined with cyclic nucleotides. Holoenzymes containing RI beta Ala and RI beta Ala/Ile gave Ka values which were higher than wild type RI beta, with the double mutant shifting toward the Ka value of RI alpha holoenzymes by about 30%. These results suggest that amino acid differences in the pseudosubstrate site may account for some, but not all, of the increased sensitivity to cyclic nucleotides exhibited by RI beta.

In situ hybridization was used to localize cAMP-dependent protein kinase (PKA) mRNAs in the adult mouse CNS. The PKA holoenzyme contains two catalytic (C) subunits and a regulatory (R) subunit dimer. Our studies demonstrate expression of two isoforms of C (C alpha and C beta) and four isoforms of R (RI alpha, RI beta, RII alpha, and RII beta) in the CNS. mRNAs for C alpha, RI alpha, and RI beta preferentially localize in the neocortex, caudate-putamen, hypothalamus, thalamus, and hippocampus. Hybridization with C beta and RII beta probes is clearly distinguished from the C alpha-like pattern by a reduced level of hybridization in the thalamus and by a relative increase in expression in the dentate gyrus compared with cell layers CA1-3 in the hippocampus. RII alpha transcripts are very specifically localized in the medial habenula. The differential expression of PKA subunit genes suggests that functional differences in cAMP responses within neural tissues may be mediated by the biochemical properties of specific PKA isoforms.

An isoform (RI beta) of the regulatory type I subunit gene of cAMP-dependent protein kinase (EC 2.7.1.37) has been characterized in mouse. The open reading frame of the RI beta cDNA is 72% identical in nucleotide sequence with the previously cloned RI gene, now referred to as RI alpha. Both genes code for a protein of 380 amino acids and their proteins are 82% identical in amino acid sequence. Sequence similarity is highest in the regions that form the pseudosubstrate-binding site of the catalytic subunit and the two cAMP binding domains. The amino-terminal portion shows the greatest dissimilarity, suggesting that the isoforms may differ in their dimerization properties or interaction with other proteins. In contrast to RI alpha, which is constitutively expressed in all tissues, RI beta is expressed in a highly tissue-specific manner. Brain and spinal cord contained significant levels of RI beta mRNA, testis RNA gave a detectable signal, and all other tissues tested were negative. Expression of a RI beta cDNA in NIH 3T3 cells resulted in the appearance of a RI subunit protein that migrated more slowly than RI alpha after NaDodSO4/PAGE. The native form of RI beta in brain could also be distinguished from RI alpha by its abnormal migration on NaDodSO4/PAGE. RI beta protein produced in 3T3 cells was shown to be functional by its ability to form a cAMP-dependent holoenzyme with the catalytic subunit.

Cyclic AMP (cAMP) and its action by way of cAMP-dependent protein kinase is important for sperm motility. Previous studies on germ cells have demonstrated a selective decrease in the amount of type I cAMP-dependent protein kinase during spermatid development, and that type II was the major form present in elongating spermatids and in mature sperm. This would indicate activation of a gene in haploid germ cells, encoding a regulatory subunit of type II protein kinase. However, haploid expression of such a gene has so far not been shown. In the present study we demonstrate high-levelled expression of a unique mRNA species for a specific regulatory subunit of type II cAMP-dependent protein kinase at late stages of spermatogenesis, i.e. during spermatid elongation.

Expression vectors were constructed that code for mutated forms of the regulatory type 1 subunit (RI) of cyclic AMP-dependent protein kinase. These mutations alter a specific amino acid which is present in each of two homologous cAMP-binding domains of the RI protein. When these expression vectors were introduced into NIH 3T3 and Y1 adrenocortical tumor cells a mutant RI protein was produced that acted in a dominant fashion to cause a 20-400-fold inhibition of cAMP-dependent protein kinase activation. In addition, processes controlled by cAMP in adrenal cells were blocked; cells became resistant to the growth-inhibitory effects of cAMP and defective in steroid synthesis. Expression of mutant RI genes in cells provides a specific means to explore the role of cAMP and protein phosphorylation in the process of intracellular signalling.

Publisher Summary This chapter focuses on the new approaches to the study of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase system with the utilization of the techniques of molecular genetics. The chapter describes the characterization of two catalytic and four regulatory subunit genes in mammalian systems. Three of these subunits—RIα, RIIα, and Cα—are constitutively expressed in most tissues although the levels of messenger RNA (mRNA) can vary significantly, most notably in male germ cells during spermatogenesis. The other three subunits—Cβ, RIβ, and RIIβ—are highly expressed in brain and are either absent or at very low levels in many other tissues. In particular, the RIIβ gene is highly inducible in several differentiating systems. The chapter further discusses the differences in functional properties of the Cα and Cβ subunits. Future work with specific antibodies should help resolve the potential for specific interactions between subtypes of R and C subunits.

We have identified and highly purified a "low Km" cAMP phosphodiesterase from bovine cardiac muscle. This phosphodiesterase was inhibited by low concentrations of cGMP and has, therefore, been temporarily designated as cGMP-inhibited phosphodiesterase. After a 16,000-fold increase in specific activity, the highly purified enzyme had a specific activity of 6 mumol/min-mg and contained three major polypeptides. Initial data indicated that all of these polypeptides were derived from a single common precursor by proteolysis. We used this enzyme preparation to generate polyclonal antisera and monoclonal antibodies directed against the "low Km" phosphodiesterase. Immunoadsorption and immunoblot analysis allowed us to identify and isolate several molecular weight species of phosphodiesterase, including a larger form than previously reported for any purified low Km phosphodiesterase. This large form of the enzyme had a subunit molecular weight of approximately 110,000 and was the only one seen in fresh extracts of cardiac muscle. Full catalytic activity was recovered in the phosphodiesterase-antibody complex and enzyme prepared by immunoprecipitation exhibited Michaelis-Menten kinetics for cAMP hydrolysis and for inhibition by cGMP. The Km for cAMP hydrolysis was 0.15 microM and the Ki for cGMP inhibition of cAMP hydrolysis was 0.06 microM. This immunoprecipitation approach also allowed us to determine that the enzyme was phosphorylated on serine residues by cAMP-dependent protein kinase, and that the low Km, cGMP-inhibited phosphodiesterase was selectively inhibited by several new cardiotonic agents. Milrinone, amrinone, and fenoximone were highly selective inhibitors of this isozyme, and the relative affinities of these inhibitors were consistent with their order of potency as positive inotropic agents. These studies suggest that the cGMP-inhibited phosphodiesterase is a receptor for several new cardiotonic drugs.

Previous studies demonstrated that both ketamine and morphine induced analgesia and catalepsy in the rat. Pre-treatment with ketamine produced cross-tolerance to morphine, whereas pretreatment with morphine did not induce cross-tolerance to ketamine but rather augmented the cataleptic response; this augmentation was attributed to residual morphine in the brain. The present studies explored the duration of the loss of righting reflex induced by sub-effective doses of ketamine and morphine, administered simultaneously. There was mutual potentiation between sub-effective doses of ketamine and morphine, but sub-effective doses of ketamine partly antagonized fully-effective doses of morphine. Latency to the loss of righting reflex, rigidity and behavior on recovery, reflected the relative predominance of ketamine or morphine in each combination. Naloxone inhibited the induced cataleptic effects. The degree and time course of development of tolerance to daily administration of sub-effective dose combinations of ketamine and morphine were similar. Rats, tolerant to ketamine-dominant combinations, were cross-tolerant to both drugs, while those tolerant to morphine-dominant combinations were cross-tolerant to morphine but showed either no cross-tolerance or an augmented response to ketamine. While the mutual potentiation, antagonism and tolerance suggest common mechanisms for the induced catalepsy, differences in latency, rigidity and behavior, asymmetry of cross-tolerance and a widely-different ID50 for naloxone would argue against an action at a single opioid site.

The effects of ketamine and morphine on pain perception and catalepsy were compared in rats. Analgesia, as measured by the latency to withdrawal of the tail from a 55 degrees C water bath (tail-flick latency difference, TFLD), was produced by both ketamine and morphine, but at widely different doses, and in each case the effect was reversed by naloxone. Catalepsy, measured by the duration of loss of righting reflex (DLRR) in catatonic animals, was induced by larger doses of both ketamine and morphine and in each case was reduced by a larger dose of naloxone. DLRR and TFLD tolerance developed rapidly and with a similar time course after daily doses of ketamine or morphine. Rats tolerant to the DLRR effect of ketamine showed cross-tolerance to morphine. Rats tolerant to the DLRR effect of morphine did not show cross-tolerance to ketamine when administered the following day; instead, these rats showed potentiation of the ketamine-induced DLRR. The degree of potentiation noted 24 hr after a single or multiple daily doses of 45 mg/kg of morphine is the same as that seen when 2 mg/kg of morphine is given simultaneously with ketamine. The residual brain level of morphine 24 hr after 45 mg/kg is similar to the level 1 hr after a 2-mg/kg dose. The augmented ketamine response in morphine-tolerant rats is postulated to be a result of residual morphine still present in the brain 24 hr after the last DLRR-inducing dose of morphine.(ABSTRACT TRUNCATED AT 250 WORDS)

Publisher Summary This chapter describes modifications of the standard techniques used for either protein kinase characterization or gene regulation studies with emphasis on the unique possibilities and pitfalls that are observed when manipulating a multisubunit enzyme system by gene transfer techniques. In utilizing the techniques, it is important to fully appreciate the complex regulatory network in which the cAMP-dependent protein kinases function. For example, the overexpression of exogenous C subunit has led to a compensatory increase in regulatory subunits in several cell types. The chapter also describe four basic parts of eukaryotic expression vectors: (1) a transcriptional promoter sequence, (2) the structural gene coding for the protein which is to be expressed, (3) a eukaryotic poly(A) addition signal sequence, and (4) a prokaryotic replication/antibiotic resistance region for growth of large amounts of the expression vector in bacteria. Expression vectors may also contain intron sequences that must be removed by RNA splicing to produce a functional mRNA but these are not essential in most cases and are not included in the protein kinase vectors.

Expression vectors were constructed that code for mutated forms of the regulatory type 1 subunit (RI) of cyclic AMP-dependent protein kinase. These mutations alter a specific amino acid which is present in each of two homologous cAMP-binding domains of the RI protein. When these expression vectors were introduced into NIH 3T3 and Y1 adrenocortical tumor cells a mutant RI protein was produced that acted in a dominant fashion to cause a 20-400-fold inhibition of cAMP-dependent protein kinase activation. In addition, processes controlled by cAMP in adrenal cells were blocked; cells became resistant to the growth-inhibitory effects of cAMP and defective in steroid synthesis. Expression of mutant RI genes in cells provides a specific means to explore the role of cAMP and protein phosphorylation in the process of intracellular signalling.