Introduction: invertebrate axons find their way.

The mechanisms that generate the immense complexity of synaptic connections within the developing nervous system have fascinated biologists for decades. Analysis of nervous system development in simple systems, such as insects, has made a major contribution to our understanding of the cellular and molecular mechanisms that control the formation of axon pathways and precise connections. This enterprise has a long, interesting, and somewhat controversial history. This collection of reviews on axon guidance in insects provides a brief update to integrate current molecular and developmental insights in a number of areas from initial axon pathfinding to the recognition of synaptic partners.

Neural development: The semantics of axon guidance.

Recent studies of the semaphorin family of axon guidance signals and their receptors have revealed a surprising versatility in the ways that they can be used solve problems in neural development, and provided new opportunities for understanding how guidance information is interpreted beneath the cell surface.

Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster.

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.

A new enhancer of position-effect variegation in Drosophila melanogaster encodes a putative RNA helicase that binds chromosomes and is regulated by the cell cycle.

In Drosophila melanogaster, position-effect variegation of the white gene has been a useful phenomenon by which to study chromosome structure and the genes that modify it. We have identified a new enhancer of variegation locus, Dmrnahel (hel). Deletion of mutation of hel enhances white variegation, and this can be reversed by a transformed copy of hel+. In the presence of two endogenous copies, the transformed hel+ behaves as a suppressor of variegation. hel is an essential gene and functions both maternally and zygotically. The HEL protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potential HEL homologues have been found in mammals, yeast and worms. HEL protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. We propose that the HEL protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that HEL is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis.

Isolation and structure elucidation of the major degradation products of cefaclor formed under aqueous acidic conditions.

The aqueous acidic degradation of the oral cephalosporin cefaclor was investigated. A number of degradation products were isolated and characterized. The degradation products can be loosely classified into three categories: thiazole derivatives, pyrazine derivatives, and simple hydrolysis or rearrangement products. Degradation pathways are proposed that involve (1) hydrolysis of the beta-lactam carbonyl with subsequent rearrangement, (2) ring contraction of the six-membered cephem nucleus to five-membered thiazole derivatives through an episulfonium ion intermediate, and (3) attack of the primary amine of the phenylglycyl side chain on the "masked aldehyde" at carbon-6 to form fluorescent substituted pyrazines.

Isolation and structure elucidation of the major degradation products of cefaclor in the solid state.

Cefaclor is a beta-lactam antibiotic that degrades slowly under normal storage conditions to several minor products. To obtain samples large enough to permit structure elucidation, cefaclor was allowed to degrade at 40 degrees C (75% relative humidity) and at 85 degrees C. The profile of degradation products formed under these conditions is qualitatively similar to the profile of degradation products observed in samples of cefaclor aged for 14 years at room temperature, although some products found in the sample degraded at 85 degrees C are not formed at the lower temperatures. Using preparative reversed-phase high-performance liquid chromatography (rp-HPLC) and a combination of spectroscopic methods, we have isolated and characterized 17 of these degradation products. Some of these products were also isolated from studies of aqueous degradations. The major products appear to have arisen from five distinct pathways: (1) isomerization of the double bond in the dihydrothiazine ring; (2) decarboxylation; (3) ring contraction of the cephem nucleus to thiazole structures; (4) oxidative attack at carbon 4 of the dihydrothiazine ring; and (5) intramolecular attack of the primary amine of the side chain on either the beta-lactam carbonyl to form 3-phenyl-2,5-diketopiperazines or the "masked aldehyde" at carbon 6 to form 2-hydroxy-3-phenylpyrazine derivatives. The pathway involving oxidation at carbon 4 is particularly important at ambient temperatures and is unique to the solid-state degradation.

Aqueous acidic degradation of the carbacephalosporin loracarbef.

The aqueous degradation of the carbacephalosporin loracarbef under moderately acidic conditions (pH range, 2.7-4.3) is described. Structures of a total of 10 compounds isolated by preparative reversed-phase HPLC have been proposed. Five of these 10 degradation compounds arose from hydrolysis of the beta-lactam ring followed by structural changes in the six-membered heterocyclic ring. Four compounds form from intermolecular reactions of loracarbef to form dimeric structures with peptide linkages. The remaining compound resulted from oxidation of the primary amine to a hydroxylamine. Pathways for the formation of these compounds from the parent loracarbef are proposed.

Isolation and structure elucidation of a novel product of the acidic degradation of cefaclor.

The acidic aqueous degradation of cefaclor, an orally administered cephalosporin antibiotic, has been investigated. The most prominent peak in the high-performance liquid chromatography profile of a degraded solution of cefaclor was isolated by preparative high-performance liquid chromatography. Mechanistically, the formation of this degradent from cefaclor involves a condensation of two cefaclor degradation products in which both products have undergone contraction from a six-membered cephem ring to a five-membered thiazole ring, presumably via a common episulfonium ion intermediate.

Expression of a Drosophila mRNA is under circadian clock control during pupation.

Rhythmic eclosion of Drosophila adults requires per gene function. We have found that a previously identified 0.9 kb RNA transcribed from DNA adjacent to per becomes abundantly expressed during pupation, just prior to eclosion. The daily synchronized emergence of young adults, coupled with a subsequent rapid decay of the transcript, is responsible for what previously appeared to be cycling of the 0.9 kb RNA in adults. In situ hybridization analyses localize the 0.9 kb transcript to the epidermis of newly eclosed adults. Conceptual translation of genomic DNA and cDNA sequences predicts that the 0.9 kb transcript produces a 261 amino acid protein containing a putative signal sequence for membrane transport at its amino terminus. Pupae that reach the same stage of development at slightly different times of day show a subsequent synchronized rise in 0.9 kb RNA levels, indicating that the expression of this transcript is under circadian clock control.

An examination of neuropeptide Y postmortem stability in an animal model simulating human autopsy conditions.

A rabbit antiserum to neuropeptide Y (NPY) was used to develop a radioimmunoassay for measuring NPY in brain tissue. By high-performance liquid chromatography, two peaks of immunoreactivity were detected in human postmortem cortex. A minor peak was seen at the void volume, while the majority of immunoreactivity comigrated with synthetic NPY standards. Using the Spokes-Koch rat model simulating human autopsy conditions, it was shown that cortical, hippocampal and striatal concentrations of NPY-like immunoreactivity are stable for up to 24 h.

A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs.

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

Membrane-bound mRNAs are recruited from preinitiated ribonucleoprotein particles in injected Xenopus oocytes.

Messenger RNA injected Xenopus oocytes exhibit a differential capacity for translation. mRNAs translated in the free cytoplasm are translated efficiently whereas mRNAs translated on the rough endoplasmic reticulum (RER) membrane are translated inefficiently. If mRNA injected oocytes are injected additionally with proteins isolated from the RER, enhanced translation of RER-bound mRNAs is observed. When examined by sucrose gradient centrifugation and RNA dot blots, most of the injected RER-bound mRNA sediments less than or equal to the 80 S monosome. The RER proteins recruit these preinitiated mRNAs onto polysomes as evidenced by a shift in sedimentation to the polysome region of a sucrose gradient. When examined by immunoblotting, the RER proteins are shown to contain a protein which reacts specifically with an antibody directed against docking protein (SRP-receptor protein). However, this putative docking protein does not appear to be the protein which actually recruits the preinitiated mRNAs onto polysomes.

Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin.

Poly(A) RNA was isolated from a rat medullary thyroid carcinoma that exhibited high levels of immunoreactive cholecystokinin (CCK). Double-stranded cDNA was synthesized from the poly(A) RNA and inserted into the Pst I site of pBR322. Bacterial colonies containing CCK cDNA were identified using the hybridization probe d(T-C-C-A-T-C-C-A-N-C-C-C-A-T-G-T-A-G-T-C). The sequence of the probe was deduced from the known amino acid sequence of porcine CCK-8, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2. The nucleotide sequence of the cDNA complementary to the mRNA of rat preprocholecystokinin was determined. The cDNA contains 33 nucleotides in the 5'-noncoding region, 199 nucleotides in the 3'-noncoding region, and 345 nucleotides coding for a precursor to CCK, which is 115 amino acids (Mr, 12,826). Examination of the rat CCK gene revealed a suggested transcriptional control sequence analogous to the "TATA" sequence located 33 nucleotides upstream from a proposed transcriptional start site. The amino acid sequence of CCK-39 is flanked by both amino-terminal and carboxyl-terminal extensions. Analysis of CCK mRNA showed that it is approximately equal to 750 nucleotides long. CCK mRNA of the rat brain and intestine appeared to be identical in size to the CCK mRNA of the carcinoma.

Somatostatin is increased in the basal ganglia in Huntington disease.

Huntington disease (HD) is an autosomal dominant hereditary disorder characterized by premature cell death, predominantly in the neostriatum. Decreased concentrations of several neurotransmitters and neuropeptides have been reported in the basal ganglia in Huntington disease. We now report that concentrations of radioimmunoassayable somatostatin are increased in extracts of the caudate (mean +/- standard error of the mean, ng/gm net weight; 247 +/- 24 versus 85 +/- 11), putamen (275 +/- 48 versus 74 +/- 11), external globus pallidus (100 +/- 10 versus 27 +/- 6), and internal globus pallidus (108 +/- 21 versus 21 +/- 8) in the disease. The concentrations of immunoreactive substance P measured in the same extracts were markedly reduced in caudate (mean +/- standard error of the mean, pmol/gm wet weight; 25 +/- 3 versus 109 +/- 20), putamen (28 +/- 7 versus 88 +/- 28), external globus pallidus (39 +/- 9 versus 196 +/- 62), and internal globus pallidus (60 +/- 17 versus 263 +/- 39), as well as in both subdivisions of the substantia nigra. Gel permeation chromatography and high-performance liquid chromatography showed radioimmunoassayable somatostatin to include peptides with physicochemical properties of the tetradecapeptide somatostatin and larger substances, including somatostatin-28-like material. A single peak of immunoreactive substance P corresponding to synthetic substance P was found by high performance liquid chromatography. These results suggest that immunoassayable somatostatin-containing neuronal elements in the neostriatum and globus pallidus in Huntington disease are affected differentially by the disease process from neurons that contain immunoreactive substance P.