Linking molecular affinity and cellular specificity in cadherin-mediated adhesion.

Many cell-cell adhesive events are mediated by the dimerization of cadherin proteins presented on apposing cell surfaces. Cadherin-mediated processes play a central role in the sorting of cells into separate tissues in vivo, but in vitro assays aimed at mimicking this behavior have yielded inconclusive results. In some cases, cells that express different cadherins exhibit homotypic cell sorting, forming separate cell aggregates, whereas in other cases, intermixed aggregates are formed. A third pattern is observed for mixtures of cells expressing either N- or E-cadherin, which form distinct homotypic aggregates that adhere to one another through a heterotypic interface. The molecular basis of cadherin-mediated cell patterning phenomena is poorly understood, in part because the relationship between cellular adhesive specificity and intermolecular binding free energies has not been established. To clarify this issue, we have measured the dimerization affinities of N-cadherin and E-cadherin. These proteins are similar in sequence and structure, yet are able to mediate homotypic cell patterning behavior in a variety of tissues. N-cadherin is found to form homodimers with higher affinity than does E-cadherin and, unexpectedly, the N/E-cadherin heterophilic binding affinity is intermediate in strength between the 2 homophilic affinities. We can account for observed cell aggregation behaviors by using a theoretical framework that establishes a connection between molecular affinities and cell-cell adhesive specificity. Our results illustrate how graded differences between different homophilic and heterophilic cadherin dimerizaton affinities can result in homotypic cell patterning and, more generally, show how proteins that are closely related can, nevertheless, be responsible for highly specific cellular adhesive behavior.

Resistance profiles of cyclic and linear inhibitors of HIV-1 protease.

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.

Synthesis and comparative molecular field analysis (CoMFA) of symmetric and nonsymmetric cyclic sulfamide HIV-1 protease inhibitors.

We have previously reported on the unexpected flipped conformation in the cyclic sulfamide class of inhibitors. An attempt to induce a symmetric binding conformation by introducing P2/P2' substituents foreseen to bind preferentially in the S2/S2' subsite was unsuccessful. On the basis of the flipped conformation we anticipated that nonsymmetric sulfamide inhibitors, with P2/P2' side chains modified individually for the S1' and S2 subsites, should be more potent than the corresponding symmetric analogues. To test this hypothesis, a set of 18 cyclic sulfamide inhibitors (11 nonsymmetric and 7 symmetric) with different P2/P2' substituents was prepared and evaluated in an enzyme assay. To rationalize the structure-activity relationship (SAR) and enable the alignment of the nonsymmetric inhibitors, i.e., which of the P2/P2' substituents of the nonsymmetric inhibitors interact with which subsite, a CoMFA study was performed. The CoMFA model, constructed from the 18 inhibitors in this study along with seven inhibitors from previous work by our group, has successfully been used to rationalize the SAR of the cyclic sulfamide inhibitors. Furthermore, from the information presented herein, the SAR of the cyclic sulfamide class of inhibitors seems to differ from the SAR of the related cyclic urea inhibitors reported by DuPont and DuPont-Merck.

Presence and localization of connexins 43 and 26 in cell cultures derived from myometrial tissues from nonpregnant and pregnant women and from leiomyomas.

OBJECTIVE: Our objective was to study the appearance and distribution of connexins 43 and 26 in various human myometrial cell cultures. STUDY DESIGN: Scrape loading, Western blotting, and immunohistochemical techniques were applied to cultured cells derived from myometrial tissues obtained from nonpregnant and pregnant women (upper and lower uterine segments) and from leiomyomas (tumor and analogous myometrial tissues). RESULTS: Scrape loading revealed the presence of metabolic coupling in all tissues. Indirect immunohistochemical studies showed membrane localization of connexin 43 in all myometrial cultures. Western blots and indirect immunohistochemical studies showed the presence and localization of the connexin 26 protein and associated gap junctions in tissues from myomas and from nonpregnant and pregnant women except for those derived from the upper segment of the pregnant uterus. CONCLUSION: These results show that human myometrial cultures express various gap junction proteins and that there are regional differences in expression of connexins in tissues from pregnant women.

Autism and hearing loss.

A group of 199 children and adolescents (153 boys, 46 girls) with autistic disorder was audiologically evaluated. Mild to moderate hearing loss was diagnosed in 7.9% and unilateral hearing loss in 1.6% of those who could be tested appropriately. Pronounced to profound bilateral hearing loss or deafness was diagnosed in 3.5% of all cases, representing a prevalence considerably above that in the general population and comparable to the prevalence found in populations with mental retardation. Hearing deficits in autism occurred at similar rates at all levels of intellectual functioning, so it does not appear that the covariation with intellectual impairment per se can account for all of the variance of hearing deficit in autism. Hyperacusis was common, affecting 18.0% of the autism group and 0% in an age-matched nonautism comparison group. In addition, the rate of serous otitis media (23.5%) and related conductive hearing loss (18.3%) appeared to be increased in autistic disorder. The study emphasizes the need for auditory evaluation of individuals with autism in order to refer those with pronounced to profound hearing loss for aural habilitation and to follow those with mild to moderate hearing loss because of the risk of deterioration.

Design and fast synthesis of C-terminal duplicated potent C(2)-symmetric P1/P1'-modified HIV-1 protease inhibitors.

An analysis of the X-ray structure of a complex of HIV-1 protease with a linear C(2)-symmetric C-terminal duplicated inhibitor guided the selection of a series of diverse target compounds. These were synthesized with the objective to identify suitable P1/P1' substituents to provide inhibitors with improved antiviral activity. Groups with various physical properties were attached to the para-positions of the P1/P1' benzyloxy groups in the parent inhibitor. A p-bromobenzyloxy compound, prepared in only three steps from commercially available starting materials, was utilized as a common precursor in all reactions. The subsequent coupling reactions were completed within a few minutes and relied on palladium catalysis and flash heating with microwave irradiation. All of the compounds synthesized exhibited good inhibitory potency in the protease assay, with K(i) values ranging from 0.09 to 3.8 nM. A 30-fold improvement of the antiviral effect in cell culture, compared to the parent compound, was achieved with four of the inhibitors. The differences in K(i) values were not correlated to the differences in antiviral effect, efficiency against mutant virus, or reduced potency in the presence of human serum. The poorest enzyme inhibitors in fact belong to the group with the best antiviral effect. The binding features of two structurally related inhibitors, cocrystallized with HIV-1 protease, are discussed with special emphasis on the interaction at the enzyme/water phase.

Tuberous sclerosis in Western Sweden. A population study of cases with early childhood onset.

OBJECTIVE: To study the prevalence of tuberous sclerosis in children and adolescents. DESIGN: Previously published diagnostic criteria for tuberous sclerosis were used. All physicians likely to encounter young patients with tuberous sclerosis were contacted by way of a screening questionnaire. SETTING: The study was performed in a circumscribed geographic area (western Sweden). PATIENTS AND OTHER PARTICIPANTS: The sample was population based. However, only patients with such severe and early symptoms that referral to a physician had been considered necessary and relatives of these patients with tuberous sclerosis could be included. This was because there is currently no diagnostic marker for tuberous sclerosis that could be used as a screening tool. RESULTS: The peak prevalence (one in 6800 individuals) for tuberous sclerosis was found in the 11- to 15-year-old age group. For the whole age cohort, 0 to 20 years, the prevalence was one in 12,900 individuals. CONCLUSIONS: The prevalence for the school-age group was the highest ever reported in the literature on tuberous sclerosis. However, it is likely that the true prevalence of tuberous sclerosis in the general population is even higher.

Autistic behaviour and attention deficits in tuberous sclerosis: a population-based study.

In a population-based sample of 28 individuals under the age of 20 years, autistic symptoms were present in 24 and DSM-III-R autistic disorder in 17. Many of the children and adolescents diagnosed as autistic also showed attention deficit/hyperactivity. There was no specific association between autistic behaviour and the presence of infantile spasms. Some of the children with tuberous sclerosis and autism were of near-normal intelligence. Indirectly, the results suggest that as many as 9 per cent of all children with autism may have tuberous sclerosis.

Glial fibrillary acidic protein in the cerebrospinal fluid of children with autism and other neuropsychiatric disorders.

The cerebrospinal fluid (CSF) of 47 children and adolescents with autism was analyzed for the contents of two astroglial proteins, the glial fibrillary acidic protein (GFA) and S 100. The results were contrasted with those obtained in similarly aged cases with other neuropsychiatric disorders (n = 25) and in normal children (n = 10). S-100 did not discriminate the groups from each other. However, GFA in autism and autistic-like conditions was at a level almost three times that in the normal group. The results could implicate gliosis and unspecific brain damage in autism. An alternative model would be increased synapse turnover regardless of underlying cause.

A sensitive ELISA for glial fibrillary acidic protein: application in CSF of children.

In the present study we describe a sensitive ELISA for determination of glial fibrillary acidic protein (GFAP). To validate the method combined determinations of GFAP and S-100 protein were performed in cerebrospinal fluid (CSF) of normal children and children with autism. The GFAP ELISA is of sandwich type and uses the biotin-avidin system. Sensitivity was 16 pg/ml. Between-day precision was 0.079 (coeff. of variance). S-100 protein concentrations were measured using a commercially available ELISA kit. Normal CSF from children and young adults were analysed. The CSF levels of GFAP in normal children were low (16-163 pg/ml). Both GFAP and S-100 protein concentrations correlated with age (P < 0.01 and P < 0.05, respectively), but the GFAP increment was more pronounced, probably reflecting the age-dependent expansion of the fibrillary astrocytes in the central nervous system (CNS). GFAP levels in children with infantile autism were higher than those in normal children of the same age range. S-100 protein concentrations were similar in both groups. High levels of GFAP in combination with normal S-100 protein concentrations in CSF indicates reactive astrogliosis in the CNS. In conclusion, the sensitive ELISA described makes it possible to measure low levels of GFAP present in the CSF of children. Combined assays of GFAP and S-100 protein can be used to discriminate between acute and chronic brain disorders in children.

Interaction between inhibitory pathways to principal cells in the lateral geniculate nucleus of the cat.

Inhibitory interactions between interneurones of the lateral geniculate nucleus (LGN) of the cat were studied with an indirect method based on intracellular recordings of synaptic responses in principal cells. Recurrent inhibitory postsynaptic potentials (IPSPs), evoked by antidromic activation of principal cell axons in the visual cortex, were depressed by a preceding stimulation of the optic tract or the visual cortex. Disynaptic feed-forward IPSPs, evoked by optic tract stimulation, were likewise depressed after cortex stimulation. The duration of the depression was in both cases about 100 ms. The effect was not due to conductance changes in the recorded principal cells or to activation of cortico-geniculate fibres. The observations indicate that perigeniculate neurones, the recurrent inhibitory interneurones of the LGN, have mutual inhibitory connexions and that they also project to intrageniculate interneurones, the inhibitory cells in the feed-forward pathway to principal cells. These conclusions were supported by intracellular recordings from a few interneurones. No evidence was found for interaction between feed-forward interneurones activated from separate eyes or for a projection from intrageniculate interneurones to perigeniculate cells. The results point to an unexpected similarity in the organization of the recurrent inhibitory system of principal cells in the LGN and of spinal motoneurones. It is suggested that the recurrent system of the LGN serves as a variable gain regulator in analogy with a recently proposed model for the spinal system.

Brain stem neurones with differential projection to functional subregions of the dorsal lateral geniculate complex in the cat.

Neurones (n = 194) in the ponto-mesencephalic reticular formation were identified as projecting to the region of the dorsal lateral geniculate nucleus, by their antidromic activation from this region. The projection of individual neurones was investigated by mapping of thresholds at closely spaced sites in the lateral geniculate nucleus and the dorsally located perigeniculate nucleus. Low thresholds, long latencies and stepwise antidromic latency shifts, occurring together, were taken to indicate termination. Low thresholds with short antidromic latencies without stepwise latency shifts were encountered more ventrally and ascribed to stimulation of larger fibres en route to terminal fields. The conduction velocity from the stimulation site of the large fibres to the cell bodies in the reticular formation was 0.9-3.1 m/s and to regions within the terminal fields 0.06-0.2 m/s. The considerable slowing of the conduction velocity, together with the extensive regions with low thresholds and the abundance of latency shifts, 0.3-19.5 ms, suggest arborization of very thin fibres within the terminal fields. Three groups of reticular neurones with different termination were distinguished: type A neurones (n = 32) terminate exclusively in the laminae of the lateral geniculate nucleus. Type B neurones (n = 22) terminate in the perigeniculate nucleus, the overlaying part of the reticular nucleus of thalamus and in the interlaminar plexuses in the lateral geniculate nucleus. Type C neurones (n = 27) terminate both in the lateral geniculate nucleus and the perigeniculate nucleus. The projection of individual neurones of all three types extended throughout their entire target nucleus/nuclei and is thus global with respect to the visual hemifield. On the basis of previous findings that stimulation in the reticular formation inhibits interneurones in both feed-forward and recurrent inhibitory pathways to relay cells in the lateral geniculate nucleus, it is suggested that the reticular neurones are inhibitory and that the type A neurones control the feed-forward inhibition, type B neurones the recurrent inhibition and type C neurones both inhibitory mechanisms together. Findings suggesting that different reticular neurones may be related to the interneuronal circuits of the X-, Y- and W-systems are discussed. Type A, B and C neurones were found intermingled, around the brachium conjunctivum and among its fibres from the level of the decussation to the posterior end of the locus coeruleus complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition from the brain stem of inhibitory interneurones of the cat's dorsal lateral geniculate nucleus.

Brain-stem control of inhibitory circuits in the dorsal lateral geniculate nucleus (d.l.g.n.) of the cat was studied with extracellular recordings from functionally identified interneurones and with intracellular recordings from principal cells. Perigeniculate neurones, the recurrent inhibitory interneurones of the d.l.g.n., were inhibited by low-threshold stimulation within a wide bilateral field of the brainstem reticular formation extending from the rostral mesencephalon to the caudal medulla oblongata. The inhibition had a latency of 10-12 ms for stimulation sites in the mesencephalon and a duration of about 100 ms. The brain-stem stimulation evoked large hyperpolarizing potentials in intracellularly recorded perigeniculate neurones, indicating that the effect was due to post-synaptic inhibition. Intrageniculate interneurones, the feed-forward inhibitory interneurones of the d.l.g.n., were inhibited with a similar time course from the same region of the brain stem. Both feed-forward and recurrent inhibitory post-synaptic potentials (i.p.s.p.s) in principal cells were depressed by a preceding stimulation of brain-stem sites effective for the interneurones. The depression had about the same time course as the inhibition of the interneurones and it occurred without a concomitant change in the membrane potential of the recorded principal cells. A small depolarizing potential, with a latency of 10-20 ms, was observed in some principal cells after brain-stem stimulation. The potential reversed polarity when i.p.s.p.s were reversed by current injection into the recorded cell indicating that it was due to disinhibition of the principal cells. The possible neuronal pathway for the inhibition of the d.l.g.n. in interneurones is considered and it is proposed that the effect is mediated by a group of neurones located in the caudal mesencephalon and the rostral pons close to the fibres of the brachium conjunctivum.

Projection of brain stem neurons to the perigeniculate nucleus and the lateral geniculate nucleus in the cat.

Small horseradish peroxidase injections in the perigeniculate nucleus (PGN) or the lateral geniculate nucleus (LGN) gave retrograde labeling of many cells in the pontomesencephalic reticular formation (RF), the nuclei raphe dorsalis and centralis linearis, locus coeruleus, nucleus of the optic tract and nucleus parabigeminalis. Antidromic stimulation was used to identify neurons in the RF projecting to the PGN-LGN complex. Threshold mapping through the PGN and the LGN shows separate projection from the reticular formation to the PGN and the LGN.