Peter Hindersson

Light and darkness regulate melanopsin in the retinal ganglion cells of the albino Wistar rat.

Circadian rhythms are daily adjusted to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Recent studies indicate that melanopsin, a newly identified opsin-like molecule, is involved in the light responsiveness of retinal ganglion cells (RGCs) constituting the RHT. In the present study, we examined the expression of melanopsin at the mRNA and protein level during a day/night cycle and during prolonged periods of light and darkness in the retina of albino Wistar rats. We observed a diurnal change in melanopsin, with mRNA level being highest at early subjective night and protein level highest at late subjective day. Prolonged exposure to darkness significantly increased melanopsin mRNA level as early as the first day, and the expression continued to increase during 5 d in darkness. The decrease in mRNA level during exposure to constant light was slower. After 48 h of light, the melanopsin mRNA level was significantly reduced, and an almost undetectable level was found after 5 d. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 d. By use of immunohistochemistry, we showed that darkness increased the amount of protein in the dendritic processes, resulting in a dense network covering the entire retina. Constant light decreased melanopsin immunostaining time dependently, beginning in the distal dendrites and progressing to the proximal dendrites and the soma. Our observations suggest that the intrinsic light-responsive RGCs adapt their expression of the putative circadian photopigment melanopsin to environmental light and darkness.

The circadian photopigment melanopsin is expressed in the blind subterranean mole rat, Spalax.

Despite severe degeneration of its eyes, the blind subterranean mole rat, Spalax, is able to adjust circadian rhythms to the environmental light/dark cycle due to a conserved retinohypothalamic tract (RHT). The photopigment mediating the circadian photoreception and it cellular localisation is unknown in the Spalax retina. Here we show, using in situ hybridization and immunohistochemistry, that melanopsin, a recently identified opsin, is expressed in retinal ganglion cells which also co-store PACAP, a neurotransmitter of the RHT. The melanopsin-component of retinal ganglion cells in the Spalax retina is well conserved resulting in a relatively higher density of melanopsin positive cells per area compared to the rat. The results show that the Spalax, as sighted animals expresses melanopsin in ganglion cells projecting to the circadian clock supporting a role of melanopsin as a circadian photopigment.

Light-induced phase shift in the Syrian hamster (Mesocricetus auratus) is attenuated by the PACAP receptor antagonist PACAP6-38 or PACAP immunoneutralization

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted (entrained) by light via the retinohypothalamic tract (RHT). The RHT contains two neurotransmitters, glutamate and pituitary adenylate cyclase‐activating polypeptide (PACAP), which are believed to mediate the phase‐shifting effects of light on the clock. In the present study we have elucidated the role of PACAP in light‐induced phase shifting at early night in hamsters and shown that (i) light‐induced phase delay of running‐wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6‐38) or by immunoblockade with a specific anti‐PACAP antibody injected intracerebroventricularly before light stimulation; (ii) PACAP administered close to the SCN was able to phase‐delay the circadian rhythm of running‐wheel activity in a similar way to light; (iii) PACAP was present in the hamster RHT, colocalized with melanopsin, a recently identified opsin which has been suggested to be a circadian photopigment. The findings indicate that PACAP is a neurotransmitter of the RHT mediating photic information to the clock, possibly via melanopsin located exclusively on the PACAP‐expressing cells of the RHT.

Cloning and nucleotide sequence comparison of the groE operon of Pseudomonas aeruginosa and Burkholderia cepacia

By alignment of GroEL amino acid sequences from four distantly related bacteria two highly conserved domains were identified. Two oligonucleotides complementary to the conserved domains were designed based on the preferred Pseudomonas aeruginosa codon usage. The primers were used in the PCR to amplify a 900‐base fragment of the P. aeruginosa groEL gene. The fragment was sequenced and the partial GroEL sequence was expanded by vectorette PCR upstream and downstream to cover the complete P. aeruginosa groE operon. The same technique was used to sequence the Burkholderia cepacia (formely Pseudomonas cepacia) groE operon and the region immediately upstream of groES. The B. cepacia groE operon is preceded by typical ‐10 and ‐35 heat shock expression signals. A total of 2041 and 2139 bp was sequenced from P. aeruginosa and B. cepacia respectively. Each revealed two open reading frames encoding two proteins with a predicted molecular mass of 10 and 57 kDa, corresponding to GroES and GroEL respectively. The GroEL proteins show an interspecies amino acid homology of 71%, and 73% with E. coli GroEL. Both GroEL proteins are 52% homologous to the corresponding human mitochondrial GroEL protein. The sequence data confirm the existence of highly conserved structures, which could be functionally important for the concerted action of GroEL and GroES in the folding and assembly of other proteins, and possibly in the initiation of autoimmune diseases.

Antigenic analysis of Pseudomonas aeruginosa and Pseudomonas cepacia GroEL proteins and demonstration of a lipopolysaccharide‐associated GroEL fraction in P. aeruginosa

Quantitative crossed immunoelectrophoresis was used to evaluate the antigenic similarity of Pseudomonas aeruginosa and Pseudomonas cepacia GroEL proteins. We found that the two proteins showed 75% identity. By using a panel of monoclonal antibodies against the P. aeruginosa GroEL protein, we identified 10 monoclonal antibodies which cross‐reacted with the P. cepacia GroEL protein and 21 monoclonal antibodies which recognized type‐specific epitopes on the P. aeruginosa GroEL protein. In crossed immunoelectrophoresis two different fractions of GroEL reactive material could be resolved. These fractions showed a reaction of partial identity. Examination of the two immunoprecipitates by Western blotting, showed that both fractions consisted of anti‐60 kDa GroEL reactive protein. One fraction, in addition, contained LPS with a characteristic ‘ladder’ reaction in modified Western blotting. We therefore conclude that this fraction represents a complex between LPS and GroEL.

Cloning and expression of the Legionella micdadei“common antigen” in Escherichia coli

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15–25 kilobase pairs (kb)) were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli ‐absorbed anti‐L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly with a monospecific rabbit antiserum raised against L. micdadei“common antigen” (CA), and an additional 13 K L. micdadei protein. The region encoding these two proteins from the 17 kb recombinant plasmid (pBA 2) was subcloned in pBGS 18+. The DNA sequence of the CA encoding region in the 2.7 kb subcloned fragment will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram‐negative species, as well as to CA structure and possible function.