Metabolic imaging of human cumulus cells reveals associations with pregnancy and live birth.

STUDY QUESTION: Can fluorescence lifetime imaging microscopy (FLIM) detect associations between the metabolic state of cumulus cell (CC) samples and the clinical outcome of the corresponding embryos? SUMMARY ANSWER: FLIM can detect significant variations in the metabolism of CC associated with the corresponding embryos that resulted in a clinical pregnancy versus those that did not. WHAT IS KNOWN ALREADY: CC and oocyte metabolic cooperativity are known to be necessary for the acquisition of developmental competence. However, reliable CC biomarkers that reflect oocyte viability and embryo developmental competency have yet to be established. Quantitative measures of CC metabolism could be used to aid in the evaluation of oocyte and embryo quality in ART. STUDY DESIGN, SIZE, DURATION: A prospective observational study was carried out. In total, 223 patients undergoing IVF with either conventional insemination or ICSI at a tertiary care center from February 2018 to May 2020 were included, with no exclusion criteria applied. PARTICIPANTS/MATERIALS, SETTING, METHODS: This cohort had a mean maternal age of 36.5 ± 4.4 years and an average oocyte yield of 16.9 (range 1-50). One to four CC clusters from each patient were collected after oocyte retrieval and vitrified. CC metabolic state was assessed using FLIM to measure the autofluorescence of the molecules NAD(P)H and FAD+, which are essential for multiple metabolic pathways. CC clusters were tracked with their corresponding oocytes and associated embryos. Patient age, Day 3 and Day 5/6 embryo morphological grades, and clinical outcomes of embryos with traceable fate were recorded. Nine FLIM quantitative parameters were obtained for each CC cluster. We investigated associations between the FLIM parameters and patient maternal age, embryo morphological rank, ploidy, and clinical outcome, where false discovery rate P-values of <0.05 were considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 851 CC clusters from 851 cumulus-oocyte complexes from 223 patients were collected. Of these CC clusters, 623 were imaged using FLIM. None of the measured CC FLIM parameters were correlated with Day 3 morphological rank or ploidy of the corresponding embryos, but FAD+ FLIM parameters were significantly associated with morphological rank of blastocysts. There were significant differences for FAD+ FLIM parameters (FAD+ fraction engaged and short lifetime) from CC clusters linked with embryos resulting in a clinical pregnancy compared with those that did not, as well as for CC clusters associated with embryos that resulted in a live birth compared those that did not. LIMITATIONS, REASONS FOR CAUTION: Our data are based on a relatively low number of traceable embryos from an older patient population. Additionally, we only assessed CCs from 1 to 4 oocytes from each patient. Future work in a younger patient population with a larger number of traceable embryos, as well as measuring the metabolic state of CCs from all oocytes from each patient, would provide a better understanding of the potential utility of this technology for oocyte/embryo selection. WIDER IMPLICATIONS OF THE FINDINGS: Metabolic imaging via FLIM is able to detect CC metabolic associations with maternal age and detects variations in the metabolism of CCs associated with oocytes leading to embryos that result in a clinical pregnancy and a live birth versus those that do not. Our findings suggest that FLIM of CCs may be used as a new approach to aid in the assessment of oocyte and embryo developmental competence in clinical ART. STUDY FUNDING/COMPETING INTEREST(S): National Institutes of Health grant NIH R01HD092550-03 (to C.R., and D.J.N.). Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. and C.R. are inventors on patent US20170039415A1. TRIAL REGISTRATION NUMBER: N/A.

Automated Measurements of Key Morphological Features of Human Embryos for IVF.

A major challenge in clinical In-Vitro Fertilization (IVF) is selecting the highest quality embryo to transfer to the patient in the hopes of achieving a pregnancy. Time-lapse microscopy provides clinicians with a wealth of information for selecting embryos. However, the resulting movies of embryos are currently analyzed manually, which is time consuming and subjective. Here, we automate feature extraction of time-lapse microscopy of human embryos with a machine-learning pipeline of five convolutional neural networks (CNNs). Our pipeline consists of (1) semantic segmentation of the regions of the embryo, (2) regression predictions of fragment severity, (3) classification of the developmental stage, and object instance segmentation of (4) cells and (5) pronuclei. Our approach greatly speeds up the measurement of quantitative, biologically relevant features that may aid in embryo selection.

Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia.

PURPOSE: This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages. METHODS: Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5-0% over the course of ~1.5 h, then 5% O2 was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration. RESULTS: Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen. CONCLUSIONS: Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

Human microtubule-associated-protein tau regulates the number of protofilaments in microtubules: a synchrotron x-ray scattering study.

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from alphabeta heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius R(MT) of MTs with increasing Phi, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in R(MT) seems to be isoform independent. Significantly, R(MT) was observed to rapidly increase for 0 < Phi < 0.2 and saturate for Phi between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C(o)(MT) of MTs leading to changes in the curvature C(MT) (=1/R(MT)). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.

Inhibition of Ca2+ release channel (ryanodine receptor) activity by sphingolipid bases: mechanism of action.

Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca2+ release channel. Three lipids, N,N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [3H]ryanodine binding by greatly increasing the rate of dissociation of bound [3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [3H]ryanodine binding. These bases also decreased the activity of the Ca2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO4 and C6 to C18 ceramides of sphingosine had no significant effect on [3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4-5 decreased the ability of the sphingolipid bases to inhibit [3H]ryanodine binding 2-3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca2+ release channel and as a class possess a common mechanism of inhibition.

Helical structure and packing orientation of the S2 segment in the Shaker K+ channel.

Six transmembrane segments, S1-S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an alpha-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane alpha helix and that the high-impact face packs against other transmembrane segments in the functional channel.

Genes and regulatory sites of the "host-takeover module" in the terminal redundancy of Bacillus subtilis bacteriophage SPO1.

Early in infection of Bacillus subtilis by bacteriophage SPO1, the synthesis of most host-specific macromolecules is replaced by the corresponding phage-specific biosyntheses. It is believed that this subversion of the host biosynthetic machinery is accomplished primarily by a cluster of early genes in the SPO1 terminal redundancy. Here we analyze the nucleotide sequence of this 11.5-kb "host-takeover module," which appears to be designed for particularly efficient expression. Promoters, ribosome-binding sites, and codon usage statistics all show characteristics known to be associated with efficient function in B. subtilis. The promoters and ribosome-binding sites have additional conserved features which are not characteristic of their host counterparts and which may be important for competition with host genes for the cellular biosynthetic machinery. The module includes 24 genes, tightly packed into 12 operons driven by the previously identified early promoters PE1 to PE12. The genes are smaller than average, with half of them having fewer than 100 codons. Most of their inferred products show little similarity to known proteins, although zinc finger, trans-membrane, and RNA polymerase-binding domains were identified. Transcription-termination and RNase III cleavage sites were found at appropriate locations.

Factors influencing [3H]ryanodine binding to the skeletal muscle Ca2+ release channel.

Optimal [3H]ryanodine binding to skeletal muscle sarcoplasmic reticulum membranes is dependent on a number of factors such as Ca2+ concentration, ionic strength, and the presence of modulators of the Ca2+ release channel. The rate of association of [3H]-ryanodine with its binding site is slower than a diffusion limited process, and often the binding reaches a peak value which is followed by a slow decline. This phenomenon makes it extremely difficult to determine kinetic constants for [3H]ryanodine binding. The inclusion of bovine serum albumin (BSA) or the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) in the incubation buffer prevents the decrease in [3H]ryanodine binding observed in association studies. BSA or Chaps slows this decline in binding partially by preventing a conversion to a more rapidly dissociating component. Pretreatment of the membranes with Chaps does not prevent the decrease in [3H]ryanodine binding, suggesting that Chaps is not exerting its effect by extracting a lipid or peripheral membrane protein. The decrease in affinity observed in the absence of BSA and Chaps appears to require the occupation of the high-affinity ryanodine binding site. Incubation for extended times in the absence of [3H]ryanodine prior to the initiation of the association produced similar curves to those obtained without preincubation. These combined results suggest that Chaps and BSA stabilize the ryanodine-modified Ca2+ release channel by preventing an alteration in the ryanodine binding site which leads to decreased affinity, thus allowing for a more quantitative interpretation of binding data.

Modulation of skeletal muscle Ca2(+)-release channel activity by sphingosine.

The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, and G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolipid also inhibited [3H]ryanodine binding to the purified ryanodine binding protein. Our results demonstrate that the inhibition of [3H]ryanodine binding by sphingosine is due to an increased rate of dissociation of bound [3H]ryanodine from SR membranes and a decreased rate of association of [3H]ryanodine to the high-affinity site. Unlike other modulators of the Ca(2+)-release channel, sphingosine can remove bound [3H]ryanodine from the high-affinity site within minutes. Sphingosine increased the rate of dissociation of [3H]ryanodine bound to a solubilized proteolytic fragment derived from the carboxy terminus of the ryanodine binding protein (cleavage at Arg4475). Sphingosine also inhibited the activity of the Ca(2+)-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direct effect of sphingosine on the Ca(2+)-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg4475 and the carboxy terminus of the Ca(2+)-release channel.

Interaction between ryanodine and neomycin binding sites on Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic reticulum (SR) calcium release. To elucidate the mechanism of inhibition, the effects of neomycin on the binding of [3H]ryanodine to the Ca2+ release channel and on its channel activity when reconstituted into planar lipid bilayer were examined. Equilibrium binding of [3H]ryanodine was partially inhibited by neomycin. Inhibition was incomplete at high neomycin concentrations, indicating noncompetitive inhibition rather than direct competitive inhibition. Neomycin and [3H]ryanodine can bind to the channel simultaneously and, if [3H]ryanodine is bound first, the addition of neomycin will slow the dissociation of [3H]ryanodine from the high affinity site. Neomycin also slows the association of [3H]ryanodine with the high affinity binding site. The neomycin binding site, therefore, appears to be distinct from the ryanodine binding site. Dissociation of [3H]ryanodine from trypsin-treated membranes or from a solubilized 14 S complex is also slowed by neomycin. This complex is composed of polypeptides derived from the carboxyl terminus of the Ca2+ release channel after Arg-4475 (Callaway, C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876-15884). The proteolytic 14 S complex isolated with ryanodine bound produces a channel upon reconstitution into planar lipid bilayers, and its activity is inhibited by neomycin. Our data are consistent with a model in which the ryanodine binding sites, the neomycin binding sites, and the channel-forming portion of the Ca2+ release channel are located between Arg-4475 and the carboxyl terminus.

Expression of estrogen receptor variants in normal and neoplastic human uterus.

Estrogen receptor variants lacking internal exons and representing dominant positive and negative activity may be involved in the initiation and/or progression of endocrine dependent tumors. To assess the role of estrogen receptor in uterine disease, we have analyzed both normal and neoplastic uterine samples for the presence of variant estrogen receptors using the sensitive technique of RT-PCR and direct automated DNA sequencing of the amplified products. Our analysis was conducted to determine the presence of spliced variants lacking exons 3 through exon 8. We demonstrate that both the normal and neoplastic human uterus contains a number of spliced variants of the estrogen receptor that co-exist with the wild type receptor. Variants lacking exons 4, 5 and 7 but not exons 3 and 6 were detected. Also, a novel partial deletion in exon 8 was detected in both the normal and neoplastic tissues, although a total deletion of this exon was not observed. In addition another region of exon 8 deletion was found to be present in one tumor tissue which also contained an insertion within this region, however, other tumors did not contain this variant. In addition, double exon deletion variants were observed lacking exons 3 and 4, exons 4 and 5, and exon 7 with part of exon 8. Although our data represents a limited number of samples it suggests that splicing of the estrogen receptor message occurs in the normal physiological setting. There does not appear to be any association between the presence or absence of spliced variants of estrogen receptor and uterine tumor formation at the mRNA level.

Effect of ryanodine on sarcoplasmic reticulum Ca2+ accumulation in nonfailing and failing human myocardium.

BACKGROUND: The purpose of this study was to determine whether abnormal Ca2+ release through ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca2+]i homeostasis that has been described in failing human myocardium. METHODS AND RESULTS: Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca2+ channels stimulates oxalate-supported, ATP-dependent Ca2+ accumulation in sarcoplasmic reticulum-derived microsomes by inhibiting concurrent Ca2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45Ca2+ accumulation in microsomes prepared from nonfailing (n = 8) and failing (n = 10) human left ventricular myocardium. In the absence of ryanodine, 45Ca2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2 +/- 6.5% increase in peak 45Ca2+ accumulation in microsomes from nonfailing hearts and a 24.3 +/- 4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [3H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. CONCLUSIONS: These results, which demonstrate a diminished stimulation of Ca2+ accumulation by ryanodine in sarcoplasmic reticulum-derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca2+]i homeostasis described in failing human myocardium.

Improved accuracy in direct automated DNA sequencing of small PCR products by optimizing the template concentration.

Data are presented illustrating the optimum concentration range of reverse transcription PCR-generated products under 500 bp for accurate base calling with direct automated DNA sequencing. A 357-bp fragment of the human estrogen receptor, which includes the DNA binding domain of the protein, was used as a representative example of a gene fragment that can be rapidly amplified and sequenced. Using the Taq DNA polymerase dye terminator sequencing protocol and automated sequencing apparatus from Applied BioSystems, 0.1 to 1.0 pmol of PCR product in a 20-microL reaction volume provided > 97% accurate base detection. Concentrations greater or lower than this range increased the number of ambiguous bases due to alterations in the signal-to-noise ratios. This procedure has been successfully utilized with 140-440-bp PCR products within the optimum concentration range. These results show that low amounts of PCR products are necessary and sufficient for direct sequence analysis.

Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.

Relationship of low affinity [3]ryanodine binding sites to high affinity sites on the skeletal muscle Ca2+ release channel.

Both high and low affinity binding sites for [3H]ryanodine exist in sarcoplasmic reticulum membranes derived from rabbit skeletal muscle. Negatively cooperative binding of [3H]ryanodine at one of four initially identical sites cannot account for some of the kinetic features of the binding to high and low affinity sites. The presence of excess unlabeled ryanodine greatly slows the rate at which [3H]ryanodine bound at the high affinity site dissociates. An examination of the rate of dissociation of [3H]ryanodine bound at increasing [3H]ryanodine concentrations reveals the existence of a second site, occupied only at high ligand concentrations. The occupation of this site correlates well with the conversion of the high affinity site from a site with a dissociation rate constant of approximately 0.0025 min-1 to one with a dissociation rate constant of less than 0.00025 min-1. The low affinity site itself has a dissociation rate constant of 0.013 min-1 and dissociation from this site is unaffected by the presence of 100 microM unlabeled ryanodine. These data suggest that the two binding sites are different but are either allosterically or sterically coupled. Association experiments support this interpretation. Low affinity binding sites for [3H]ryanodine exist in transverse tubule (t-tubule) as well as sarcoplasmic reticulum membranes. High concentrations of both ryanodine and ruthenium red inhibit the binding of [3H]PN200-110 to the dihydropyridine-binding protein in t-tubule membranes. Whether the low affinity site in t-tubule membranes is related to that found in sarcoplasmic reticulum membranes is not yet known.

Hormonal regulation of the mRNA for cysteine-rich intestinal protein in rat jejunum during maturation.

Cysteine-rich intestinal protein (CRIP) has been implicated as an important zinc-binding protein in the rat intestine. However, its specific role remains undefined. As an approach to the ultimate elucidation of the function of CRIP, we have explored the role of glucocorticoids and L-thyroxine (T4) in the increase of CRIP mRNA that occurs during postnatal development. Hydrocortisone administration on day 10 elicited a precocious increase of CRIP mRNA. The response to hydrocortisone was readily detectable 12 h after injection. Lack of endogenous glucocorticoids in rat pups adrenalectomized on day 9 impeded but did not prevent the normal rise of CRIP mRNA. Furthermore, injections of dexamethasone (DEX) on days 10, 16, and 18 led to a loss of responsiveness of CRIP mRNA as the pups matured. The administration of T4 alone resulted in a small increase of CRIP mRNA, whereas when combined, T4 and DEX synergistically raised the concentration of CRIP mRNA. All of these patterns of response to hormone manipulation indicate the possibility that CRIP is a mediator of glucocorticoid action on the developing intestine. They do not appear to support the hypothesis that CRIP plays a role in zinc transport during the postnatal period. The potent effects of glucocorticoids and T4 on CRIP mRNA levels should provide useful tools for further investigations in this area.

A novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene.

A cDNA representing a unique Ca2+/calmodulin-dependent protein kinase has been cloned and sequenced from a rat brain cDNA library. This enzyme, expressed in brain, testis, and spleen, is only 32% identical to the various isoforms of Ca2+/calmodulin-dependent protein kinase II. The sequence of the COOH-terminal 169 amino acids is identical to that of a previously described male germ cell-specific calmodulin-binding protein called calspermin (T. Ono, G.R. Slaughter, R.G. Cook, and A.R. Means, J. Biol. Chem. 264:2081-2087, 1989). This identity extends to the nucleic acid sequence and includes all but the first 130 nucleotides of the calspermin cDNA. Primer extension and sequence of a genomic fragment containing the unique calspermin sequence reveals that this mRNA is derived from the kinase transcription unit by germ cell-specific use of a unique exon. In situ hybridization was used to demonstrate that both kinase and calspermin mRNAs are expressed during spermatogenesis. The kinase mRNA is first detected in early meiotic cells and declines to a low level in haploid cells. Calspermin mRNA first appears in pachytene primary spermatocytes and continues to increase as cells complete meiosis and undergo terminal differentiation. These results show that differential utilization of a single gene during spermatogenesis is used to generate mRNAs that encode proteins with distinct functions.

Regulatory and structural motifs of chicken gizzard myosin light chain kinase.

The amino acid sequence for chicken smooth muscle myosin light chain kinase (smMLCK) was deduced from a full-length cDNA. This has allowed definition of both the complete sequence of the inactive 64-kDa proteolytic fragment, which contains the pseudosubstrate autoregulatory sequence, and of the active 61-kDa Ca2+/calmodulin-independent fragment, which lacks the autoregulatory domain. Comparison of the two sequences shows that the autoregulatory domain extends from Asn-780 to Arg-808. The peptide Leu-774 to Ser-787 does not inhibit smMLCK, whereas peptides of similar or shorter length from the pseudosubstrate region (Ser-787 to Val-807) are potent inhibitors. These data define the autoregulatory region as being contained within and probably identical to the pseudosubstrate domain. The catalytic and regulatory regions are flanked by several copies of 100-amino acid segments containing one of two consensus motifs. These motifs are absent from mammalian skeletal muscle MLCK or from Dictyostelium discoideum MLCK but are present in the Caenorhabditis elegans unc-22 gene product and the titin molecule of skeletal muscle myofibrils. These results indicate that the amino acid sequence of smMLCK encodes multiple functional motifs in addition to the catalytic domain.

Treatment of patients with advanced cancer using multiple long-term cultured lymphokine-activated killer (LAK) cell infusions and recombinant human interleukin-2.

Escalating doses of recombinant human interleukin-2 (rIL-2) were combined with long-term cultured rIL-2 activated killer cells to treat patients with disseminated melanoma, renal cell cancer, and colon cancer. Twenty-four patients were entered, 12 with renal cell cancer, 8 with colon cancer, and 4 with melanoma; 23 were evaluable for efficacy and toxicity. The (dose-related) toxicities were moderate to severe and consisted of fever, chills, rigors, weight gain, hypotension, mild confusion, elevation of liver enzymes and serum creatinine, thrombocytopenia, and eosinophilia. No cardiac events (arrhythmias or myocardial infarction) were recorded. None of the patients were admitted to the intensive care unit, and no deaths occurred. Two partial responses were observed, one at relatively low doses of rIL-2 in a patient with renal cell carcinoma and one at the highest dose level in a patient with malignant melanoma. The maximally tolerated dose level of rIL-2 for this study was 6 X 10(6) U/m2 i.v./day. The recommended dose for further studies is 3 X 10(6) U/m2 i.v./day in three divided doses.

Inhibitors of sterol synthesis. Chemical synthesis, structure, and biological activities of (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one, a metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one.

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is a potent inhibitor of sterol synthesis with significant hypocholesterolemic activity. (25R)-3 beta,26-Dihydroxy-5 alpha-cholest-8(14)-en-15-one (II) has been shown to be a major metabolite of I after incubation with rat liver mitochondria. Described herein is the chemical synthesis of II from diosgenin. As part of this synthesis, improved conditions are described for the conversion of diosgenin to (25R)-26-hydroxycholesterol. Benzoylation of the latter compound gave (25R)-cholest-5-ene-3 beta,26-diol 3 beta,26-dibenzoate which, upon allylic bromination followed by dehydrobromination, gave (25R)-cholesta-5,7-diene-3 beta,26-diol 3 beta,26-dibenzoate. Hydrogenation-isomerization of the delta 5.7-3 beta,26-dibenzoate to (25R)-5 alpha-cholest-8(14)-ene-3 beta,26-diol 3 beta,26-bis(cyclohexanecarboxylate) followed by controlled oxidation with CrO3-dimethylpyrazole gave (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one 3 beta,26-bis(cyclohexanecarboxylate). Acid hydrolysis of the delta 8(14)-15-ketosteryl diester gave II. 13C NMR assignments are given for all synthetic intermediates and several major reaction byproducts. The structure of II was unequivocally established by X-ray crystal analysis. II was found to be highly active in the suppression of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured mammalian cells and to inhibit oleoyl coenzyme A-dependent esterification of cholesterol in jejunal microsomes.