Heat-shock proteins during growth and sporulation of Bacillus subtilis

Four major heat-shock proteins (hsps) with apparent molecular masses of 84, 69, 32 and 22 kDa were detected in exponentially growing stationary phase and sporulating cells of Bacillus subtilis heat-shocked from 30 to 43 degrees C. The most abundant, hsp69, is probably analogous to the E. coli groEL protein. These proteins were transiently inducible by heat-shock. Partial purification of RNA polymerase revealed several other minor hsps. One of these, a 48 kDa polypeptide probably corresponds to sigma 43. The synthesis of this polypeptide and at least two other proteins appeared to be under sporulation and heat-shock regulation and was affected by the SpoOA mutation.     

Intracellular Distribution of Heat Shock Proteins in Pigeon Pea (Cajanus cajan)

The proteins synthesized In response to higher temperature In pigeon pea (Cajanus cajan) plants have been studied with respect to their Intracellular localization using root tissue. The heat shock proteins (hsps) of 18, 20, 22 and 24 kD were found to be associated with mitochondrial and membrane fractions, while the 60, 70 and 81 kD hsps were found In the soluble fraction. No evidence for the presence of hsps among the proteins synthesized in organello by isolated mitochondria could be obtained. Low molecular weight hsps (18, 20, 22 and 24 kD) were found associated with mitochondria Isolated from the heat shocked tissue suggesting that these hsps may have been transported post-translationally into mitochondria.     

Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis

Heat shock proteins (hsps) are a group of highly conserved polypeptides found in a wide variety of organisms. Polypeptides of sporozoites and merozoites of Eimeria bovis, blotted onto nitrocellulose, were probed with antibodies to artificially constructed peptides representing portions of the cDNA-generated fragment of pf75, the 75K hsp of merozoites of Plasmodium falciparum. Polypeptide antigens of sporozoites and merozoites of E. bovis with molecular weights of 46K, 71-72K, and 75K reacted with antibodies against pf75, indicating that they are hsp70 (the 70K family of hsps) or hsp70 cognates (noninducible proteins homologous to hsps). Radiolabeling with 125I and treatment with antibodies against pf75 detected a 71K antigen on the merozoite surface. Hsps in sporozoites of E. bovis are either constitutive or evoked by treatment at 37 C for in vitro excystation. If hsp70 is mandatory for parasite survival, it may prove to be an appropriate antigen for a vaccine against bovine coccidiosis.     

Heat Shock Proteins of Pigeon Pea (Cajanus cajan)

The proteins synthesized in response to higher temperature ina tropical legume-plant pigeon pea (Cajanus cajari) have beenstudied using polyacrylamide gel electrophoresis. Ten to twelveheat shock proteins (hsps) of molecular weights ranging from15 to 81 kDa are synthesized by excised roots when temperatureis raised by 10?C above their normal growth temperature (30?C).The heat shock response is rapid and the presence of hsps canbe detected just 30 min after raising the temperature. Hspscan not be seen in stained gels, and their presence can onlybe monitored by fluorography. The results indicate the transientnature of their synthesis. Most of the hsps are of nuclear origin,however, at least two of them, 18 and 60 kDa proteins appearto be synthesized in mitochondria. (Received September 2, 1987; Accepted February 3, 1988)     

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.     

Cryoprotection provided by heat shock treatment in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells exposed to 43 degrees C (normal being 30 degrees C) exhibit the synthesis of heat shock proteins (hsps). Time course studies indicated that the major hsps (97 kDa, 85 kDa and 70 kDa family) are induced within 10 min. of heat shock and attain maximum amount with two hours of treatment. The viability of cells decreased by 99% when directly frozen into liquid nitrogen. However, a prior heat shock (2 hours) increased the cell survival by 20-30 fold. Such an effect of prior heat shock treatment could be supported by light and electron microscopical studies. Differential scanning calorimetric analysis of whole cells revealed that heat shock treatment decreases the denaturation (delta H) of total cellular proteins. A direct correlation between the degree of hsp inducibility and protection against freezing and thawing injury was observed. Cycloheximide treatment curtailed the synthesis of hsps as well as protection against subsequent freezing. This suggests that prior heat shock treatment protects the cells from freezing injury and, furthermore, that hsps can act as biological cryoprotectants.     

Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins

Bovine seminal plasma (BSP) contains a family of major proteins designated BSP-A1/A2, BSP-A3, and BSP-30kDa (collectively called BSP proteins) that bind to sperm at ejaculation and potentiate sperm capacitation. Homologous proteins have been identified in stallion, boar, goat, and ram seminal plasma. We report here the isolation and characterization of homologous proteins from bison seminal vesicle secretions. Seminal vesicle secretory proteins were precipitated by adding cold ethanol and recovered by centrifugation. The precipitates were resuspended in ammonium bicarbonate, dialyzed, and lyophilized. Lyophilized proteins were dissolved in 0.05 M phosphate buffer (PB) and loaded onto a gelatin-agarose column. The unadsorbed proteins and adsorbed proteins were eluted with PB and 5 M urea in PB, respectively. The gelatin-adsorbed fraction was analyzed by SDS-PAGE and revealed the presence of four major proteins designated BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, and BiSV-28kDa (BiSV: bison seminal vesicle proteins). Heparin-Sepharose chromatography allowed the separation of BiSV-16kDa, which did not bind heparin from other BiSV proteins, which bound heparin. Immunoblotting revealed that BiSV-16kDa cross-reacted with BSP-A3 antibodies, BiSV-17kDa and BiSV-18kDa cross-reacted with BSP-A1/-A2 antibodies, and BiSV-28kDa cross-reacted with BSP-30kDa antibodies. Radioimmunoassays indicated that approximately 25% of bison seminal vesicle total proteins are related to BSP proteins. The amino-terminal sequencing indicated that BiSV proteins share almost 100% sequence identity with BSP proteins. In addition, BiSV proteins bind to low-density lipoproteins isolated from hen's egg yolk. These results confirm that BSP protein homologs are present in mammalian seminal plasma and they may share the same biological role.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

Heat shock proteins of chicken lens

The presence of heat shock proteins HSP-40, HSP-70, and HSc-70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP-40, HSP-70, and HSc-70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP-40, HSP-70, and HSc-70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunofluorescence technique. Data obtained from these experiments revealed that HSP-40, HSP-70, and HSc-70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP-40, HSP-70, and HSc-70 can interact with protein of these areas and protect them from stress induced denaturation.     

Heat shock proteins and chilling sensitivity of mung bean hypocotyls

Excised mung bean (Vigna radiata L.) hypocotyl sections wereexposed to 40 C for up to 4 h in the presence or absence of50 µM cycloheximide (CHX) before being held at a non-chilling(20 C) or chilling (2.5 C) temperature. Mung bean hypocotyltissue is chilling sensitive, and the rate of solute leakageis highly correlated with the extent of chilling injury. A 3h heat shock at 40 C reduced chilling-induced solute leakageby up to 40%, but leakage was similar to non-heat-shocked hypocotylswhen CHX was present. Specific proteins were labelled when hypocotylswere exposed to [³⁵S] methionine during the last hour of heatshock. The nine most intense bands on the autoradiographs ofSDS-PAGE gels of extracted protein corresponded to molecularweights of 114, 79, 73, 70, 60, 56, 51, 46, and 18 kDa. The18 kDa band reached a maximum after 1 h at 40 C and then rapidlydecreased in intensity as the heat shock continued, becomingundetectable at 4 h. The four most intense bands after 3 h at40 C corresponded to molecular weights of 79, 70, 51, and 46kDa. The synthesis of these four hsps was markedly reduced whenthe hypocotyl sections were exposed to CHX during heat shock.During chilling for 6 d, the levels of hsps 79 and 70 remainedsignificantly higher in tissue that was heat shocked prior tochilling than in tissue that was not heat shocked. In contrast,the levels of hsps 51 and 46 were similar in bothheat-shockedand control tissues. Heat-shock-induced chilling tolerance waslost between 6 and 9 d ofstorage at 2.5 C; this loss coincidedwith the decay of hsps 79 and 70 to control levels. These resultssuggest that heat shock induces an increase in both chillingtolerance and the de novo synthesis of specific heat shock proteins;namely hsps 79 and 70. This is the first report showing a relationshipbetween heat-shock-induced chilling tolerance and specific heat-shock-inducedproteins. Key words: Ion leakage, protein synthesis, Vigna radiata     

Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in mouse alpha-crystallin gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.     

Preparation and characterization of polyclonal antibodies specific for the 69 and 100 k-dalton forms of human 2-5A synthetase

Recently, the existence of 40-, 46-, 69- and 100- kDa forms of 2',5'-oligoadenylate (2-5A) synthetase have been established in interferon-treated human cells. Using monoclonal antibodies specific for 69- and 100- kDa forms of 2-5A synthetase, we purified these proteins by immunoaffinity chromatography and raised murine polyclonal antibodies. All immunized mice developed antibodies (anti-69 or anti-100 kDa form) which were characterized by their capacity to immunoprecipitate [35S] cysteine labeled proteins from interferon-treated cells or identify these proteins by electrophoretic transfer immunoblot analysis of extracts from control and interferon-treated cells. The 69 and 100 kDa 2-5A synthetases were induced in different types of human cells, such as Daudi, BJAB, HeLa and differentiated HL-60 cells. These enzymes were not detectable nor induced in MRC5 and undifferentiated HL-60 cells.     

High temperature induces the synthesis of heat-shock proteins and the elevation of intracellular calcium in the coral Acropora grandis

 Elevated temperatures induce coral bleaching. To investigate subcellular changes in heat-stressed corals, we examined heat-shock proteins (hsps) and the concentration of calcium in coral cells at elevated temperatures. After heat treatment, three hsps with approximate molecular weights of 70, 60 and 35 kDa were prominent. The 35 kDa heat-shock protein was further demonstrated to be heme oxygenase by immunoblotting, suggesting the involvement of oxidative stress in coral cells during heat treatment. Since the expression of hsps needs the activation of a calcium signal, the change of intracellular calcium concentration in coral cells was examined by a FURA 2 fluorescence method under heat treatment. Intracellular calcium concentration increased at high temperatures with or without the presence of ambient calcium. The extent of the calcium concentration increase was constant for at least 6 hours. This result indicates the existence of an active calcium signal in coral cells at elevated temperatures. Accepted: 4 August 1996     

Identification of a novel GPI-anchored CRISP glycoprotein,MAK248, located on the posterior head and equatorial segment of cynomolgus macaque sperm

To identify a sperm-surface component that is highly antigenic, we immunized female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored sperm surface proteins that were released following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Five different adjuvants were used in combination with the PI-PLC-released proteins, and three of these proteins (24, 48, and 53 kDa) were shown to be potent antigens for immunization of female monkeys. The 53 kDa protein was found to be a surface coating protein and not a GPI-anchored protein. Polyclonal antibodies to the 24 kDa protein and the 48 kDa protein were produced in rabbits. The two antibodies recognized both proteins on Western blots. The same rabbit antibodies recognized 28, 18, and 10 kDa bands on a Western blot of chemically reduced PI-PLC-released proteins, suggesting that the 48 kDa protein is a dimer of the 24 kDa protein, which we refer to as MAK248. Rabbit polyclonal antibodies developed to reduced fragments of the 24 kDa protein showed that the 18 and 10 kDa bands are proteolytic peptide fragments of the 24 kDa protein. Screening of tissues from male macaques showed that MAK248 is expressed only in the epididymis. Microsequencing of two proteolytic fragments of the 18 kDa component showed 100% amino acid homology to a 233 deduced amino acid sequence previously identified in human testes genome. Antibodies to MAK248 recognized a 24 kDa protein released from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized the equatorial segment and posterior head regions of capacitated cynomolgus macaque sperm. Structural analysis suggests that MAK248 is a novel CRISP protein and a member of the CAP (CRISP, Ag 5, PR-1) family of proteins. Based on amino acid sequence homology, it is possible that MAK248 functions as a protease inhibitor.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.