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Abstract: The present study examined the effects of ovariectomy and subsequent estradiol replacement on learning in young adult rats using a set of instrumental avoidance paradigms differing in the nature and extent of prior experience in the learning context. Thus, one group of animals was placed directly into avoidance learning (AV). A second group was trained on an appetitive task first, and then transferred into the aversive context (AP-AV). The third group was exposed to the training context without any specific appetitive response requirement, and then required to learn an active avoidance response (Context-AV). We found that estradiol (OVX+E) impaired avoidance acquisition in all cases relative ovariectomized controls (OVX). In contrast, while avoidance learning is improved following appetitive training or context exposure in both OVX+E and OVX animals, the OVX+E animals profit to a greater extent from the appetitive or context experience than do the OVX controls. We suggest that this difference may be due to enhanced attentional processes or improved hippocampal processing of contextual factors. Thus, estradiol negatively influences simple associative avoidance learning in ovariectomized rats, but appears to promote positive transfer.

Abstract: Rats with selective lesions of the nucleus basalis magnocellularis (NBM) and sham-lesion control animals were tested in an operant appetitive-to-aversive transfer task. We hypothesized that NBM lesions would not affect performance in the appetitive phase, but that performance would be impaired during subsequent transfer to the aversive phase of the task. Additional groups of NBM lesion and control rats were tested in the avoidance condition only, where we hypothesized that NBM lesions would not disrupt performance. These hypotheses were based, on the argument that the NBM is not necessary for simple association learning that does not tax attention. Both the appetitive phase of the transfer task and the avoidance only task depend only on simple associative learning and are argued not to tax attention. Consequently, performance in these tasks was predicted to be spared following NBM lesions. Complex, attention-demanding associative learning, however, is argued to depend on the NBM. Performance, in the aversive phase of the transfer task is both attentionally demanding and associatively more complex than in either the appetitive or aversive tasks alone; thus, avoidance performance in the NBM lesion group was predicted to be impaired following transfer from prior appetitive conditioning. Results supported our hypotheses, with the NBM lesion group acquiring the appetitive response normally, but showing impaired performance following transfer to the aversive conditioning phase of the transfer task. Impairments were not attributable to disrupted avoidance learning per se, as avoidance behavior was normal in the NBM lesion group tested in the avoidance condition only.

Substituted guanines and pyrimidines were tested as inhibitors of cyclin B1/CDK1 and cyclin A3/CDK2 and soaked into crystals of monomeric CDK2. O⁶-Cyclohexylmethylguanine (NU2058) was a competitive inhibitor of CDK1 and CDK2 with respect to ATP (Ki values:  CDK1, 5 ± 1 μM; CDK2, 12 ± 3 μM) and formed a triplet of hydrogen bonds (i.e., NH-9 to Glu 81, N-3 to Leu 83, and 2-NH2 to Leu 83). The triplet of hydrogen bonding and CDK inhibition was reproduced by 2,6-diamino-4-cyclohexylmethyloxy-5-nitrosopyrimidine (NU6027, Ki values:  CDK1, 2.5 ± 0.4 μM; CDK2, 1.3 ± 0.2 μM). Against human tumor cells, NU2058 and NU6027 were growth inhibitory in vitro (mean GI50 values of 13 ± 7 μM and 10 ± 6 μM, respectively), with a pattern of sensitivity distinct from flavopiridol and olomoucine. These CDK inhibition and chemosensitivity data indicate that the distinct mode of binding of NU2058 and NU6027 has direct consequences for enzyme and cell growth inhibition.

Tyrosinase is a copper-containing enzyme that regulates melanin biosynthesis in mammals. Mutations at a single N-glycosylation sequon of tyrosinase have been reported to be responsible for oculocutaneous albinism type IA in humans, characterized by inactive tyrosinase and the total absence of pigmentation. To probe the role that each N-glycosylation site plays in the synthesis of biologically active tyrosinase, we analyzed the calnexin mediated folding of tyrosinase N-glycosylation mutants. We have determined that four of the six potential N-glycosylation sites, including that associated with albinism, are occupied. Analysis of the folding pathway and activity of 15 tyrosinase mutants lacking one or more of the occupied N-glycosylation sites shows that glycans at any two N-glycosylation sites are sufficient to interact with calnexin and give partial activity, but a specific pair of sites (Asn(86) and Asn(371)) is required for full activity. The mutants with less than two N-glycosylation sites do not interact with calnexin and show a complete absence of enzyme activity. Copper analysis of selected mutants suggests that the observed partial activity is due to two populations with differential copper content. By correlating the degree of folding with the activity of tyrosinase, we propose a local folding mechanism for tyrosinase that can explain the mechanism of inactivation of tyrosinase N-glycosylation mutants found in certain pigmentation disorders.

The three‐dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti‐trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5′‐AAT‐3′ region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen‐bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen‐bonded contact with an amidinium nitrogen atom, N3 of the 5′ end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

The current techniques for X‐ray‐diffraction data collection from macromolecular crystals at cryogenic temperatures are reviewed. The development of the experimental methods is outlined and the basic concepts pertaining to radiation damage and cryoprotection are summarized. Emphasis is placed on the practical aspects important to the success of the techniques, and a detailed account of these is presented.

The three‐dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti‐trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5′‐AAT‐3′ region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen‐bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen‐bonded contact with an amidinium nitrogen atom, N3 of the 5′ end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain. The kinase domain of the catalytic subunit has been expressed inEscherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9). A three-fold excess of magnesium to ATP was used for crystal growth. The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1° to 0.3°. The crystals are orthorhombic, space groupP212121with unit cell dimensionsa=47.1 A˚,b=69.1 A˚,c=112.9 A˚ and one molecule per symmetric unit. Data to 3 A˚ resolution have been collected and structure determination is in progress.

This study focuses on consumers' level of knowledge, opinions, and degree of preparedness concerning selected practices and regulations of the funeral industry. Questionnaires were returned by 75 percent of the sample of faculty and staff at a large university. Opinions of respondents rejected the status quo concerning various practices and regulations of the funeral industry, indicating a desire to support newly suggested changes in the industry. Preparedness scores indicated that almost 9 out of 10 of the respondents were quite unprepared for their own funerals. The mean achievement score regarding knowledge as measured by the Consumer Funeral Test, which was developed for this study, was 4.0, representing 57 percent correct. Significant statistical relationships existed between knowledge scores and the age, education, and sex of the respondents; those who were older and had more education scored higher, and men scored higher than women. No relationships existed, however, among knowledge, opinion, and preparedness scores and the demographic variables of age, education, income, sex, and religion, raising the fundamental question of the value of funeral information programs for consumers.

Sialidases (EC 3.2.1.18 or neuraminidases) remove sialic acid from sialoglycoconjugates, are widely distributed in nature, and have been implicated in the pathogenesis of many diseases. The three-dimensional structure of influenza virus sialidase is known, and we now report the three-dimensional structure of a bacterial sialidase, from Salmonella typhimurium LT2, at 2.0-A resolution and the structure of its complex with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid at 2.2-A resolution. The viral enzyme is a tetramer; the bacterial enzyme, a monomer. Although the monomers are of similar size (approximately 380 residues), the sequence similarity is low (approximately 15%). The viral enzyme contains at least eight disulfide bridges, conserved in all strains, and binds Ca2+, which enhances activity; the bacterial enzyme contains one disulfide and does not bind Ca2+. Comparison of the two structures shows a remarkable similarity both in the general fold and in the spatial arrangement of the catalytic residues. However, an rms fit of 3.1 A between 264 C alpha atoms of the S. typhimurium enzyme and those from an influenza A virus reflects some major differences in the fold. In common with the viral enzyme, the bacterial enzyme active site consists of an arginine triad, a hydrophobic pocket, and a key tyrosine and glutamic acid, but differences in the interactions with the O4 and glycerol groups of the inhibitor reflect differing kinetics and substrate preferences of the two enzymes. The repeating "Asp-box" motifs observed among the nonviral sialidase sequences occur at topologically equivalent positions on the outside of the structure. Implications of the structure for the catalytic mechanism, evolution, and secretion of the enzyme are discussed.

THERE have been several recent reports of the detection of 14C in modern carbon samples using large van de Graaff accelerators1–3and cyclotrons4as mass spectrometers. The attraction of using such systems is the possibility that very small samples (∼5 mg) might be quickly and accurately dated. We have considered the design criteria for a mass spectrometer incorporating a tandem van de Graaff accelerator to be built specifically for carbon dating and in this letter we present data which show that the measurements are possible using much smaller and simpler systems than those discussed previously.

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.