Dynamics of confined water molecules.

We present femtosecond midinfrared pump-probe measurements of the molecular motion and energy-transfer dynamics of a water molecule that is enclosed by acetone molecules. These confined water molecules show hydrogen-bond and orientational dynamics that are much slower than in bulk liquid water. This behavior is surprising because the hydrogen bonds to the C=O groups of the acetone molecules are weaker than the hydrogen bonds in bulk water. The energy transfer between the O-H groups of the confined water molecules has a time constant of 1.3 +/- 0.2 ps, which is >20 times slower than in bulk water. We find that this energy transfer is governed completely by the rate at which hydrogen bonds are broken and reformed, and we identify the short-lived molecular complex that forms the transition state of this process.

Vibrational relaxation and coupling of two OH-stretch oscillators with an intramolecular hydrogen bond.

We studied the vibrational dynamics of the OH-stretch oscillators of an alcohol with two vicinal OH groups using femtosecond midinfrared pump-probe spectroscopy. The absorption spectrum of pinacol (2,3-dimethyl-2,3-butanediol) in CDCl3 shows two OH-stretch peaks belonging to hydrogen bonded and free OH groups. The anharmonicities of the hydrogen-bonded and free OH-stretch vibrations are 180 and 160 cm(-1), respectively. The lifetime T1 of the OH-stretch vibration is found to be 3.5 +/- 0.4 ps for the hydrogen bonded and 7.4 +/- 0.5 ps for the free OH group. We observed sidebands in the transient spectra after excitation of the bonded OH group, which we attribute to a progression in a low-frequency hydrogen-bond mode. The sideband is redshifted 60 cm(-1) with respect to the 0 --> 1 transition. Due to the coupling between the two OH groups and the presence of the sidebands, simultaneous excitation of both OH-stretch vibrations leads to oscillations on the pump-probe signal with frequencies of 40 and 60 cm(-1).

Photophysics and optical switching in green fluorescent protein mutants.

We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable "one-color" systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in "color" on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species.

Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning.

Several studies have led to the conclusion that, in the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a photoconversion involving excited-state proton transfer occurs from an A- to a B-form, while an intermediate I-form was held responsible for the green fluorescence. Here we have identified the I-form of wild-type GFP in absorption, located the 0-0 transitions of all three forms A, B and I, and determined vibrational frequencies of the ground and excited states. The intrinsically narrow 0-0 transitions are revealed by the wavelengths at which holes can be burnt. The pathways of photointerconversion are unraveled by excitation, emission and hole-burning spectroscopy. We present an energy-level scheme that has significant implications for GFP-mutants, which likewise can occur in the three photo-interconvertible forms.

Purification of bovine thyroid-stimulating hormone by a monoclonal antibody.

A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by [3H]thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH.

Selective depletion of substance P-immunoreactive neurones in the transitional zone of the colon in piebald lethal mice.

The distribution of substance P in the colon of piebald lethal (s(1)/s(1)) mice was studied by radioimmunoassay and immunohistochemistry. These animals inherit as a Mendelian recessive trait an aganglionic distal colon. In the region proximal to the aganglionic segment, there is an extensive transitional or hypoganglionic zone, in which the total number of nerve cells in the myenteric plexus is reduced, while those in the submucous plexus tend to be normal. Immunohistochemical studies indicate that substance P-immunoreactive neurones accounted for approx. 10% of the total number of normal myenteric neurones, but in the hypoganglionic region they accounted for about 5%, and this difference was statistically significant. By radioimmunoassay, the concentrations of substance P in both the aganglionic and the hypoganglionic regions of the colon were reduced compared with the corresponding segments in normal mice. However calculation of the mean substance P content per neurone revealed similar quantities (about 1 fmol) in both normal and s(1)/s(1) mice. Substance P-immunoreactivity in the tissue extracts eluted in the same position as the synthetic peptide on ion exchange and gel filtration chromatography. It is suggested that a sub-population of substance P-immunoreactive neurones in the hypoganglionic zone is selectively depleted compared with other myenteric neurones. The factors involved remain to be elucidated, but this strain of mice could prove useful for studies of the mechanisms involved in differentiation and development of enteric peptidergic neurones.

Immunohistochemical studies on the gastrointestinal tract using antisera to Met-enkephalin and Met-enkephalin Arg6Phe7.

Antisera specific for the C-terminus of Met-enkephalin and two of its variants isolated from adrenal medulla and brain, namely Met-enk Arg6 and Met-enk Arg6Phe7, have been used in immunohistochemical studies of the gastrointestinal tract in rat, mouse and guinea pig. Met-enk and Met-enk Arg6Phe7-like immunoreactivities were found with similar distribution in nerve cell bodies of the myenteric plexus, and in fibers that were particularly dense in the myenteric plexus and circular smooth muscle. The Met-enk Arg6 antiserum showed weak staining of nerve cells and fibers. In rat and mouse, the antiserum to Met-enk Arg6Phe7, but not those to Met-enk or Met-enk Arg6, also stained numerous endocrine-like cells of the antral mucosa. These were identified as gastrin cells by elution and re-staining experiments with C-terminal gastrin antisera. The rat and mouse gastrin cells might conceivably express the enkephalin gene, but fail to process it to yield Met-enkephalin; alternatively, the Met-enk Arg6Phe7-like immunoreactivity in gastrin cells could be due to a cross-reacting peptide that does not belong to the opioid series.

Regional differences in the development of cholecystokinin-like activity in rat brain.

The postnatal development of cholecystokinin (CCK) in rat brain was studied by radioimmunoassay and bioassay of tissue extracts. Marked differences were found in the patterns of development in different regions of the brain. In the cerebellum and brainstem of newborn rats the concentrations of CCK8-like immunoreactivity were 40-100% those in adults, whereas in more rostral regions the concentrations were 1-10% of those in adults. Between 0 and 14 days in concentrations of CCK-like activity measured by radioimmunoassay increased up to 30-fold in hypothalamus, cortex and olfactory bulb; in the cortex there were further increases up to 42 days. Cortical CCK was also measured by bioassay on rabbit gall bladder in vitro; bioactivity was identified in foetuses, and after birth showed a similar pattern of increase to that measured by radioimmunoassay. Immunoreactive material in extracts of neonatal cerebellum, brainstem and cortex was identified as CCK8 on the basis of cross-reactivity with different antisera, and chromatographic properties on gel filtration. The results raise the possibility of different rates of maturation of central CCKergic systems.

A rational approach to the fixation of peptidergic nerve cell bodies in the gut using parabenzoquinone.

Study of the possible chemical mechanisms by which parabenzoquinone acts as a fixative in the localisation of neuronal peptides by immunohistochemistry suggested several possible improvements to standard techniques. In particular, by using favourable conditions for regenerative oxidation reactions, i.e. fixing at 37 degrees C in well oxygenated, slightly alkaline buffer, it was possible to improve the localisation of substance P-, vasoactive intestinal polypeptide-, enkephalin- and somatostatin-like immunoreactive peptides such that nerve cell bodies were routinely visualised without the use of drugs to block axonal transport. Application of the improved fixation method is likely to facilitate study of the organisation of peptidergic systems.